Increased NT-proANP predicts risk of congestive heart failure in Cavalier King Charles spaniels with mitral regurgitation caused by myxomatous valve disease

Increased NT-proANP predicts risk of congestive heart failure in Cavalier King Charles spaniels with mitral regurgitation caused by myxomatous valve disease

Journal of Veterinary Cardiology (2014) 16, 141e154 www.elsevier.com/locate/jvc Increased NT-proANP predicts risk of congestive heart failure in Cav...

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Journal of Veterinary Cardiology (2014) 16, 141e154

www.elsevier.com/locate/jvc

Increased NT-proANP predicts risk of congestive heart failure in Cavalier King Charles spaniels with mitral regurgitation caused by myxomatous valve disease Anders S. Eriksson, DVM, PhD a,b,*, ¨ggstro ¨m, DVM, PhD c, Jens Ha Henrik Duelund Pedersen, DVM, DrVetSci d,g, Kerstin Hansson, DVM, PhD c, ¨rvinen, DVM, PhD a, Anna-Kaisa Ja Jari Haukka, PhD e, Clarence Kvart, DVM, PhD

f

a

Department of Equine and Small Animal Medicine, University of Helsinki, Box 57, 00014 Helsinki, Finland b Minerva Institute for Medical Research, Biomedicum Helsinki 2U, Tukholmankatu 8, 00290 Helsinki, Finland c Department of Clinical Sciences, Swedish University of Agricultural Sciences, Box 7054, S-750 07 Uppsala, Sweden d The Royal Veterinary and Agricultural University, Copenhagen, Denmark e Faculty of Medicine, Hjelt Institute, University of Helsinki, Box 40, 00014, Finland f Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Box 7011, S-750 07 Uppsala, Sweden Received 13 November 2012; received in revised form 8 May 2014; accepted 9 May 2014

KEYWORDS Natriuretic peptide; Endothelial dysfunction; Chronic valvular

Abstract Objectives: To evaluate the predictive value of plasma N-terminal proatrial natriuretic peptide (NT-proANP) and nitric oxide end-products (NOx) as markers for progression of mitral regurgitation caused by myxomatous mitral valve disease. Animals: Seventy-eight privately owned Cavalier King Charles spaniels with naturally occurring myxomatous mitral valve disease.

* Corresponding author. E-mail addresses: [email protected], [email protected] (A.S. Eriksson). g Current address: Novo Nordisk A/S, Novo Nordisk Park, 2760 Ma ˚løv, Denmark. http://dx.doi.org/10.1016/j.jvc.2014.05.001 1760-2734/ª 2014 Elsevier B.V. All rights reserved.

142 disease; Dog

A.S. Eriksson et al. Methods: Prospective longitudinal study comprising 312 measurements over a 4.5 year period. Clinical values were recorded, NT-proANP concentrations were measured by radioimmunoassay, and NOx were analyzed colorimetrically. To predict congestive heart failure (CHF), Cox proportional hazards models with time-varying covariates were constructed. Results: The hazard ratio for NT-proANP (per 1000 pmol/l increase) to predict future CHF was 6.7 (95% confidence interval, 3.6e12.5; p < 0.001). The median time to CHF for dogs with NT-proANP levels >1000 pmol/l was 11 months (95% confidence interval, 5.6e12.6 months), compared to 54 months (46 e infinity) for dogs with concentrations 1000 pmol/l (p < 0.001). Due to intra- and inter-individual variability, most corresponding analyses for NOx were insignificant but dogs reaching CHF had a lower mean NOx concentration than dogs not reaching CHF (23 vs. 28 mmol/l, p ¼ 0.016). Risk of CHF increased with increase in heart rate (>130 beats per minute) and grade of murmur (3/6). Conclusions: The risk of CHF due to mitral regurgitation is increased in dogs with blood NT-proANP concentrations above 1000 pmol/l. Measurement of NT-proANP can be a valuable tool to identify dogs that may develop CHF within months. ª 2014 Elsevier B.V. All rights reserved.

Abbreviations ACEI

angiotensin converting enzyme inhibitor AIC Akaike’s information criterion cGMP cyclic guanosine monophosphate CHF congestive heart failure CI confidence interval CKCS Cavalier King Charles spaniels eNOS endothelial nitric oxide synthase HPLC high-performance liquid chromatography HR hazard ratio LA/Ao left atrium to aortic root ratio MMVD myxomatous mitral valve disease MR mitral regurgitation NO nitric oxide NOx nitric oxide end-products, nitrite and/or nitrate NT-proANP N-terminal pro-A-type (or atrial) natriuretic peptide NT-proBNP N-terminal pro-B-type (or brain) natriuretic peptide NYHA New York Heart Association SD standard deviation SVEP Scandinavian Veterinary Enalapril Prevention study

Introduction Mitral regurgitation (MR) caused by myxomatous mitral valve disease (MMVD) is the most common cardiac disease affecting dogs.1 Mitral regurgitation

progresses slowly, but dogs living long enough often develop congestive heart failure (CHF). Although the stages2 and progression3,4 of disease are well described, tools to predict deterioration and onset of CHF are sparse. Several biomarkers, particularly the natriuretic peptides, have been evaluated to help identify dogs at risk before or at the time of CHF. The biologically inactive but stable N-terminal fragments of pro-A (atrial) and pro-B-type (brain) natriuretic peptides (NT-proANP and NT-proBNP, respectively) have been suggested as useful biomarkers for this purpose.5 NT-proANP levels increase with severity of MR, and dogs in CHF have up to 7 times higher mean plasma concentrations of NT-proANP than normal dogs.6 Natriuretic peptides have shown potential to distinguish between cardiac and respiratory disease7,8 and to predict CHF.7,9e12 However, reports on the power of natriuretic peptides to discriminate between earlier disease stages of MMVD are more disperse.6,12e14 One reason for this disparity is the use of different clinical classification schemes. Another may relate to dissimilarities in statistical methods applied. As an example, cross-sectional methods compare means (or ranks) between groups and are not designed to detect individual trends over time. Moreover, age15 and decreased renal function,16e18 both of which may affect plasma levels of natriuretic peptides, may confound results. To reduce the impact of these factors this study uses longitudinal analyses that are able to account for time-varying covariates and individual trends over time.19 Nitric oxide (NO) is a soluble gas produced locally by vascular endothelial cells. The effect of NO is

NT-proANP predicts risk of congestive heart failure in mitral regurgitation mediated via the same secondary messenger, cyclic guanosine monophosphate (cGMP), as for natriuretic peptide signaling and NO can be measured as the soluble metabolites nitrate and nitrite (NOx).15 Under normal circumstances endothelial nitric oxide synthase (eNOS) produces NO to induce vasodilation and inhibit aggregation of thrombocytes.20 In hypoxia and inflammation, however, the inducible form (iNOS) produces NO in excess. Together with oxygen radicals, NO may then form toxic peroxynitrites. In the progression of heart failure NO may therefore mediate either essential vasodilation or cell death, via its toxic effects.21,22 In healthy dogs plasma NOx levels correlate with NT-proANP concentrations and increases with age.15 Indices of increased tissue activity have been reported in the mitral valves in dogs with MMVD,23 and signs of endothelial dysfunction have been observed in dogs with MMVD through necropsy material,24,25 by peripheral blood flow measurements,26,27 and through analysis of plasma cGMP or precursors28 and end-products29 to NO. However, to our knowledge, there are no data on changes and trends in endothelial function with regard to the progression of MMVD. We hypothesized that an increase in NT-proANP and NOx could be revealed by a longitudinal analysis using time-varying covariates in dogs with MR caused by MMVD and that these data may predict future CHF. The primary objective was to evaluate changes in plasma concentration of NT-proANP and NOx with the progression of MMVD in dogs. Secondary objectives were to assess the relationships of NT-proANP and NOx to clinical variables such as age, heart rate and heart murmur.

Materials and methods All together, 312 examinations in 78 dogs were performed in a prospective, longitudinal, multicenter study with a surveillance time of up to 4.5 years. Client-owned Cavalier King Charles spaniels (CKCS) were enrolled at the Veterinary University Hospitals in Finland, Sweden and Denmark. Dogs were a subset of the previously published Scandinavian Enalapril Prevention (SVEP) study.30 Dogs were randomly allocated to treatment with enalapril (0.25e0.5 mg/kg daily) or to placebo.30 The Ethical Committees in each of the respective countries approved the protocol and consent was obtained from all owners involved in the study.

Animals Only CKCS afflicted with MR attributable to MMVD in a modified New York Heart Association (NYHA)

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functional class I or II were enrolled. Thus, all dogs included had a characteristic MR murmur verified by echocardiography, some had enlarged left atrium and ventricle, but none had past or present clinical or radiographic signs of cardiac failure.30 A complete blood count and a screening biochemistry profile were performed. Dogs with treatment for heart failure within 2 months before inclusion, signs of other systemic disease and dogs outside the weight range of 5e15 kg were excluded. All dogs participating in the SVEP study enrolled at the university hospitals in Finland, Sweden, and Denmark were included in this substudy without additional inclusion or exclusion criteria.

Study outline At entry and every 12 months (3 days) thereafter, dogs underwent a clinical examination, thoracic radiography, plasma NT-proANP and NOx assay. In addition, baseline left atrium to aortic root ratio (LA/Ao) was determined at entry.31 The end-point event was diagnosis of onset of CHF. Dogs that did not develop CHF during the study period were censored in analyses. In addition to these scheduled annual examinations, dogs were examined in the same manner whenever the owner suspected signs of CHF. If CHF could not be verified based on preset criteria, the dog continued in the trial. To ensure uniform grading of heart murmurs and reading of thoracic radiographs, researchers met prior to and annually during the trial for blinded auscultation and radiography reading sessions. The diagnosis of CHF was made if dogs had clinical signs of congestion (cough, dyspnea and/or nocturnal restlessness) and exercise intolerance (minimum of two out of four). In addition, dogs had to show signs of subjectively evaluated cardiomegaly including left atrial enlargement,32 and cardiogenic pulmonary edema on thoracic radiographs. To minimize risk of false positive diagnoses dogs continued in the trial until signs could be confirmed. In case of true congestive signs, owners requested a new evaluation in a very short time. At the completion of the trial all radiographs were reevaluated by a radiologist (KH) in a blinded fashion. If CHF was not confirmed, the initial diagnosis of heart failure was to be rejected.

Measurement of biomarkers Blood samples for measurement of NT-proANP and NOx were drawn in chilled tubes containing 0.5 ml 0.15 M ethylenediaminetetraacetate (EDTA). Plasma was then separated in a cooled centrifuge

144 and stored at 80  C. The proper storage of NTproANP blood samples was determined earlier.33,34 Samples collected during the first 2 years were analyzed during the trial and those collected thereafter were analyzed at the end of the trial. We used a direct radioimmunoassay (RIA) for NT-proANP measurement as described previously.6,15,35 Briefly, antibodies were raised in rabbits against human proANP-(79e98), which is identical to the corresponding sequence in the dog, except for amino acid number 95. NT-proANP was determined directly from plasma. The within and between assay coefficients of variation were <10% and <15%, respectively. Gel filtration highperformance liquid chromatographic (HPLC) analyses of plasma samples consistently showed the presence of only one immunoreactive species corresponding in size to that of intact NT-proANP [proANP-(1e98)]. Plasma nitrite and nitrate (NOx) were analyzed with a colorimetric method.15 Briefly, nitrate (NO3) was enzymatically converted to nitrite (NO2), Griess reagents were added and absorbance (l ¼ 560 nm) measured. Standard curves with added known concentrations of NO3 coincided with expected values.

Statistical analyses Comparisons between continuous variables in either two or several groups with even distribution were made by Student’s t-test or analysis of variance (ANOVA), respectively, and a Welch test was employed if variances were unequal. Repeated measures were analyzed by a multivariable ANOVA with visits (time) as contrasts. For non-normal distributions and categorical data, nonparametric tests (Wilcoxon or KruskalleWallis and Chi-square tests, respectively) were applied. Analyses were performed using the JMPh software package and statistical significance was defined as p < 0.05. Hazard ratios (HRs) were analyzed in Cox models36 in the free statistics software R.i The ordinary Cox model was extended to control for individual change of variables by time (i.e. individual trajectories were analyzed).37 The surveillance time (survival) was the time between entrance of study to CHF or censoring. The variation of NT-proANP was modeled using a mixed effect model with time as a fixed covariate and dog as a random component.38 The effect of time and change in HR with level of a variable was modeled using the spline technique h

JMP v. 9, SAS Institute Inc., Cary, NC, USA. R version 2.15, 2012, The R Foundation for Statistical Computing, ISBN 3-900051-07-0. i

A.S. Eriksson et al. as described previously.39 Since some dogs were also examined at times between these scheduled annual visits, whenever owners suspected signs of CHF, the ordinary Cox’s model could not handle these different intervals. In contrast to the fixed covariate (i.e. variable), the time-varying covariate refers to a variable that is not necessarily constant through the whole study but may have different values at different time points. Therefore two main models were constructed, one evaluating hazards of baseline values (baseline HR) and another assessing the HR with change in time and measured values of respective variable (longitudinal HR)37,39 using the survival package19 in R.i A Cox’s proportional-hazard analysis with a counting process approach was used with CHF as the end-point and the end of follow-up as censoring time.37 With this counting process approach, an individual may have both time-fixed and time-varying explanatory variables. The follow-up time for each individual was divided into periods, in which time-varying variable values were constant. That is, if the value of a time varying variable at baseline is 0, and changes after one year to 1, then this individual has two data rows. The first row with entry time “baseline”, exit time “one year” and variable value 0; and a second row with entry time “one year”, exit time (i.e. censoring or event time), and variable value 1. The center for examination, treatment and gender were used as fixed variables and the following as time varying variables e age, weight, NT-proANP, NOx, heart murmur, and heart rate. Results are reported as hazard ratios (HR) with 95% confidence intervals (CI). The HR expresses the difference between two hazard functions. That, in turn, is the relative risk for individuals with a certain level or type of predictor (i.e. covariate) compared to another type. In multivariable models all predictors with a univariable p-value <0.20 were entered in the model. Predictors were excluded from the model one at a time for p-values 0.10. Since correlated predictors may be excluded in the backwards selection process, each predictor was separately reintroduced in final models to check for significance. The goodnesses-of-fit for alternative multivariable models were assessed by comparing the difference in likelihood ratios in a Chi-square test. Models differing in predictors (e.g. for NOx) were compared by Akaike’s Information Criterion (AIC), which penalizes for every increase in the number of variables. The KaplaneMeier method was used to estimate the median time to endpoint for each group and plot time to event curves. A cut-off value for the KaplaneMeier groups was based on a graphical evaluation of the risk profile

NT-proANP predicts risk of congestive heart failure in mitral regurgitation of NT-proANP and interval HRs. The last visit before the end of the trial (for NT-proANP values under chosen threshold), either censoring or CHF, or the first visit with an NT-proANP exceeding a chosen threshold was used. A log-rank test with right censoring was used to determine whether a significant difference existed between groups.

Results Baseline characteristics are described in Table 1. Thirty-four dogs reached the endpoint (CHF) in the survival analysis. None of the dogs primarily diagnosed with CHF were later reclassified as censored in the blinded evaluation of thoracic radiographs. Our results show that dogs with plasma NT-proANP concentrations 1000 pmol/l survived significantly longer before the onset of CHF than dogs with NTproANP concentrations >1000 pmol/l (Fig. 1). Seventy-six out of 78 dogs were eligible for KaplaneMeier analysis in which the cut-off value of 1000 pmol/l was chosen based on change in risk and interval HR models. Only one dog had a plasma concentration of NT-proANP >1000 pmol/l at inclusion. At the last visit, 31 out of 34 dogs in CHF had an NT-proANP concentration above 1000 pmol/ l and 38 out 41 censored dogs had measured values 1000 pmol/l. Specifically, observed median time to CHF for all dogs with NT-proANP levels >1000 pmol/l was 6 months. The KaplaneMeier survival model analysis showed that median time to CHF for dogs with NT-proANP 1000 pmol/l was longer (54 months; 95% CIs, 46 e infinity months) than for dogs with plasma levels above 1000 pmol/l (11 months; 95% CIs, 9e19 months, p < 0.0001) (Fig. 1). Table 1 Baseline characteristics in 78 dogs with myxomatous valvular disease (MMVD). Gender F/M Age (years) Weight (kg) Time in study (months) Heart rate (beats/min) Heart murmur (L/M/H) Cardiomegaly (Y/N) LA/Ao NT-proANP (pmol/l) NOx (mmol/l)

35/43 6.6  2.1 10.0  1.8 31.4  14.7 128  22 10/23/5 42/36 1.42  0.22 465  228 19.4  9.8

Continuous variables reported as mean  standard deviation. L, low intensity murmur (grades 1 and 2); M, moderate intensity (grades 3 and 4); H, high intensity murmurs (grades 5 and 6); LA/Ao, left atrial to aortic root dimension; NTproANP, N-terminal-pro A-type natriuretic peptide; NOx, end-products (nitrate and nitrite) of endothelial nitric oxide production.

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In univariable Cox models (Table 2) heart rate and heart murmur were the only significant predictors of CHF in both the baseline and the longitudinal model but NT-proANP was a highly significant predictor in the longitudinal model. Heart rate, heart murmur and NT-proANP were all independent predictors of CHF in multivariable models (Table 3). Gender, treatment or center did not affect any outcome evaluated (all p > 0.2). Remarkable changes in HR by level of continuous predictors were observed in the splinesmoothed graphical models (Fig. 2). This means the risk does not increase or decrease linearly with an increase of the predictor. It may even first decrease and then increase, as shown for NOx (Fig. 2, Panel C). The interval HR analysis (Table 4) confirmed the graphical impression (Fig. 2), that risk of heart failure is increased for NT-proANP concentrations over 1000 pmol/l. Consistent with results of the univariable and multivariable Cox models, all intervals for heart murmur (compared to grade 1e2/6) and heart rate (compared to heart rate <115 beats/min), except the interval 115e130 beats/min, were significant. The median NT-proANP value at the end of the study for dogs reaching CHF was 1516 (interquartile range, 1158e1871) pmol/l. Corresponding values for dogs that did not reach CHF (i.e. censored) was 672 (interquartile range, 400e830) pmol/l, p < 0.0001). Figure 3 presents smoothed regression lines (Panel A) of NT-proANP for dogs both reaching and not reaching CHF before the end of study. The real trajectories in time are presented in panels B and C (dogs not reaching and reaching CHF, respectively). Panel A shows that average NT-proANP levels increase faster starting from 2 years before failure. The time scale in these graphs counts backwards from the endpoint/censor time. In all 112 NOx samples were analyzed. The HR for NOx followed a bell curve with increased risk of heart failure both for low and high values (Fig. 2, Panel C). In the interval Cox regression model, NOx levels between 18.5 and 35 mmol/l were associated with a significantly lower risk of CHF (Table 4). The reason for this awkward risk profile may be that dogs with unaltered endothelial function have the ability to increase their endothelial cell NO production to maintain blood flow with decreasing pump function. However, high concentrations indicate either progression of disease or inflammation, i.e. activation of the detrimental inducible nitric oxide synthase (iNOS) pathway. NOx did not reach significance in either the baseline or the longitudinal Cox model, nor were there significant

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A.S. Eriksson et al.

Figure 1 KaplaneMeier plot of the 78 dogs in the study as a function of time to congestive heart failure or right censoring. Dogs with plasma levels of N-terminal fragments of pro-A (atrial) natriuretic peptides (NT-proANP) above 1000 pmol/l experienced significantly shorter median times to event (11 months; interquartile range, 9.0e18.8 months) than dogs with NT-proANP levels maintained 1000 pmol/l (54 months; interquartile range, 45.6 months e infinity), p < 0001.

associations of NOx with other variables including NT-proANP, age, gender, grade of heart murmur or heart rate, but dogs which developed CHF had a lower mean plasma level of NOx than dogs not reaching CHF (censored) during the study period (23 vs. 28 mmol/l, p ¼ 0.016). This difference was not affected by treatment with enalapril. In the repeated measures analysis, test differences between the last annual visit before and the end of trial were significant (19.5  3.1 vs. 29.7  2.5, p ¼ 0.015, p for whole model ¼ 0.03) indicating that the average production of nitric oxide increased with time.

Discussion The study showed an increased risk of CHF (HR ¼ 6.7, p < 0.001) for dogs with MMVD as blood NT-proANP levels increased above 1000 pmol/l. Specifically, dogs with plasma NT-proANP concentrations above 1000 pmol/l reached CHF in a significantly shorter median time than dogs with NTproANP below 1000 pmol/l (11 vs. 54 months, p < 0.0001). Increases in heart murmur grade and heart rate were also identified as risk factors [HR ¼ 2.28 and 1.29 (for 10 unit increase in heart rate), p < 0.001 and 0.019, respectively]. Unfortunately, in this study, nitric oxide, measured

as nitrates and nitrites (NOx), had high variability and the sample size lacked power to predict future CHF. Taken together, these results indicate that measurement of NT-proANP can be a valuable tool in the clinical setting to identify dogs that may develop CHF within months.

Natriuretic peptides Importantly, our findings add temporal as well as between-subject-variation dimensions to earlier cross-sectional studies8,12 reporting an increase in plasma levels of NT-proANP and NT-proBNP in severe MR, and especially in CHF. If the individual variation in a variable is high, as it is for natriuretic peptides, standard cross-sectional statistical tests lose power, because there is no way to estimate the individual time course of the specific variable. However, in this study, we extended the ordinary Cox proportionals hazards model to enable control for time-varying variables and between-subject variation. Since all dogs in this study had MMVD, NT-proANP levels were expected to overlap with dogs reaching CHF vs. not reaching CHF (censored). The observed median time to CHF of 6 months is near the median time of 4.6 months between the two last visits observed in the PREDICT-study.10 However, their estimation was based on a

NT-proANP predicts risk of congestive heart failure in mitral regurgitation

147

Table 2 Hazard ratios (HR) for values at baseline [A] and HRs for the same variables in the extended longitudinal Cox model [B]. Variable

A. Baseline Coefficient

Age (years) Weight (kg) Gender (M/F) Treatment (P/E) Heart rate (per 10 beats/min) Heart murmur NT-proANP (per100 pmol/l) NOx (mmol/l)

B. Longitudinal

HR

CI

p-value

0.107 0.119 0.145 0.133 0.019

1.11 1.13 1.16 1.14 1.21

0.96e1.29 0.94e1.35 0.60e2.24 0.59e2.20 1.01e1.45

0.15 0.21 0.69 0.72 0.031

0.698 0.088

2.01 1.01

1.56e2.59 0.86e1.19

0.002 0.92

0.92

0.83e1.02

0.11

0.084

Coefficient

HR

CI

p-value

0.115 0.021 0.145 0.133 0.026

1.12 1.02 1.16 1.14 1.29

0.97e1.30 0.87e1.19 0.60e2.24 0.59e2.20 1.13e1.48

0.13 0.79 0.69 0.72 0.019

0.826 0.906

2.28 1.21

1.71e3.05 1.14e1.29

<0.001 <0.001

0.015

0.98

0.91e1.06

0.70

coeff

The hazard ratio (HR) is calculated as e , where coeff. is the coefficient in the Cox regression formula returning the HR for an increase in one unit of a variable. The baseline model utilizes only baseline values (i.e. an ordinary Cox model). The longitudinal model (extended Cox model) utilizes all measured values from start (baseline) to last value before end (i.e. heart failure or censoring). For grouping variables (Treatment and Gender) the two models are identical, because variables do not change by time. CI ¼ 95% confidence interval; E, enalapril and P, placebo; NT-proANP, N-terminal pro A-type natriuretic peptide; NOx, degradation products of nitric oxide measured as nitrate and nitrite.

dichotomized time scale and only true observations. To visualize estimated time to CHF on a continuous scale, we plotted KaplaneMeier survival curves (Fig. 1). In this model the median time to CHF for dogs with NT-proANP levels >1000 pmol/l was 11 months, which is considerably longer than in the PREDICT study, but resembles the survival to CHF in dogs with elevated levels of NT-proBNP in an other report.9 However, that study included dogs on treatment, had a predefined follow-up period of 6 months at which survival was surveyed, but excluded dogs surviving 1e180 days, as well as survivors with mild azotemia. As such, the study design is too different from ours for meaningful comparisons.

Table 3 Multivariable extended Cox regression models taking into account change of variable by time. Model A

c

Bc Cd

Predictors a

d

NT-proANP Heart Murmur NT-proANPa Heart Rateb Heart Murmur Heart Rateb

HR

95% CI

p-value

1.13 1.86 1.17 1.26 2.17 1.29

1.08e1.29 1.32e2.62 1.10e1.24 1.11e1.41 1.65e2.93 1.14e1.45

0.007 <0.001 <0.001 <0.001 0.003 <0.001

HR, hazard ratio; CI, confidence interval. a Per 100 pmol/l increase in N-terminal pro A-type natriuretic peptide. b Per 10 beats/minute increase in heart rate. c Model A better than model B (p ¼ 0.036). d Model C better than Model B (p ¼ 0.008), difference between A and C not significant.

To the best of our knowledge, there is no longitudinal study comparing the superiority of NTproBNP to NT-proANP for predicting the outcome in MMVD dogs. Theoretically, plasma concentrations of NT-proANP may rise earlier than NTproBNP, because MR caused by MMVD increase LA pressure causing stretch, which induce early secretion of ANP by atrial myocytes with later shift to ventricles in cardiac overload.40 In contrast, BNP is primarily synthesized by ventricular myocytes, although BNP in the dilated heart can also be synthesized in the atria.40 Indeed, both natriuretic peptides correlate with severity of regurgitation in MMVD dogs, but only NT-proBNP was related to left ventricular end-diastolic dimensions.12 It has also been shown that MMVD dogs (N ¼ 115) in different disease stages have significant differences in plasma (C-terminal) ANP concentrations.14 However, molecular heterogeneity of the circulating forms of natriuretic peptides causes a serious risk of preanalytical error in assays for NT-proBNP and, to a lesser extent, NT-proANP.41 The most robust and reliable assays use antibodies directed at the central portions of NT-proANP or NT-proBNP. Therefore, in order to assess the superiority of these natriuretic peptides, novel validated canine immunoassays should be used and testing should be performed in prospective longitudinal clinical studies using predefined standardized protocols, sufficient sample size, and using appropriate statistical methods. Contrary to the longitudinal model, the baseline HR for NT-proANP was not significant (HR ¼ 1.01,

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A.S. Eriksson et al. p ¼ 0.92; Table 2). This finding may seem contradictory. However, because NT-proANP levels rise late in the progression of disease, as indicated by the short time to CHF (Fig. 1), it was only possible to predict the onset of CHF within months of it occurring. A change in the hazard rate, i.e. increase in hazard (Fig. 2), improved the discriminatory and predictive value of NT-proANP (Table 4). The hazard rate for NT-proANP increased steeply near 1000 pmol/l. The increase in hazard rate then stabilized after 1000 pmol/l (Fig. 2). Therefore the difference in HR between a plasma concentration of 600 to 1000 pmol/l versus 1000 to <1500 was evident (HR compared to NT-proANP <600 ¼ 1.6 and 9.4, respectively), whereas a step to the next interval (1500 pmol/l) was small (HR ¼ 11, Table 4). The overall implementation to the clinical setting could be to measure blood NT-proANP whenever signs of cardiac enlargement are suspected or confirmed on radiography or echocardiography. If plasma levels are below 600 pmol/l, MR is moderate and there is only minor left sided enlargement, the dog is unlikely to be at risk of CHF. If plasma levels are 600e1000 but there is evident left sided enlargement, there is a need for intensified followup and, if plasma concentration of NT-proANP exceeds 1000 there may, in addition, be a need for medication. The rationale behind this conclusion is that high plasma concentrations of natriuretic peptides indicate that adaptive mechanisms are maximally activated, after which the dog may need external diuretics or other supportive treatment.

Age

Figure 2 Hazard plots on a logarithmic scale showing hazard of continuous variables NT-proANP (Panel A), age (Panel B), and NOx (Panel C) relative to their absolute value on a linear scale. Because hazards are not described as horizontal lines the Cox regression proportionality assumption is violated for these variables. NT-proANP ¼ N-terminal fragments of pro-A (atrial) natriuretic peptides; NOx ¼ metabolic end-products (nitrate and nitrite) of endothelial nitric oxide (NO).

Without special constructs, Cox regression models assume linearity (proportionality) with time, meaning that the effect of a predictor is constant. However, our models could account for a change, and we present spline-smoothed estimates of variation in HR by absolute value of a predictor (Fig. 2). This method of analysis and graphical presentation facilitates finding clinically meaningful intervals for HR analyses (Table 4). As mentioned previously, the risk of CHF with increases in NT-proANP was not linear (Fig. 2, Panel A). Specifically, age did not independently increase risk of CHF in the longitudinal Cox model (p ¼ 0.13, Table 2), although the risk for dogs aged 7e9 years was increased (HR ¼ 2.16, p ¼ 0.037, Table 4). This is in part consistent with a reported increase in risk for dogs over 8 years of age.3 However, in our material, after 7e9 years, the risk declined (Fig. 2, Panel B). This may seem

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Table 4 Hazard ratios (HR) for fitted intervals of continuous variables in interval Cox regression models. All intervals are compared with the reference interval. Variable

Coefficient HR for interval (CI) p-valuea p-valueb

Reference interval

Interval

NT-proANP (pmol/l)

0e<600

Age (years)

0e<5.5

NOx (mmol/l)

0e<9

Heart rate (beats/min)

0e114

Heart murmur (grade 1e6)

1e2

600e1000 1000e<1500 1500 5.5e<7 7e<9 9 9e<18.5 18.5e<35 35 115e<130 130e<150 150e<185 185 3e4 5e6

0.48 2.24 2.40 1.24 2.16 1.60 2.18 3.06 1.88 0.54 1.90 1.64 2.53 2.25 2.82

1.6 9.4 11.0 3.5 8.6 5.0 0.11 0.05 0.15 1.7 6.7 5.1 12.5 9.5 16.7

(0.59e4.4) (4.2e21.6) (3.9e30.9) (0.40e30.3) (1.14e65.5) (0.59e41.3) (0.008e1.7) (0.004e0.55) (0.012e1.92) (0.44e6.7) (2.5e17.5) (1.7e16.1) (2.4e65) (2.9e30.6) (3.9e71.6)

0.350 <0.001 <0.001 0.260 0.037 0.139 0.110 0.015 0.150 0.440 <0.001 0.005 0.003 <0.001 <0.001

<0.001

0.023

0.140c

<0.001

0.007

HR, hazard ratio; CI, confidence interval; NT-proANP, N-terminal pro A-type natriuretic peptide. a p-value by likelihood ratio test for each interval. b p-value for whole model. c p < 0.05 by Wald and logrank tests.

peculiar because not only would one expect older dogs be at higher risk, because of deteriorating endothelial function and compensatory mechanisms, but also because of faster progression of valve degeneration. The interpretation of the finding, however, is not straightforward. First, to detect an age-dependent effect, the age structure should be sufficiently spread, which was not the case for our material. Second, the oldest dogs affected by MMVD may die for reasons other than CHF and are therefore lost from the analysis (censored) resulting in a reduced risk in old dogs. That is in fact what happened, as demonstrated in Fig. 2, Panel B. However, the prevalence of MMVD increases with age,42 but this increase is not the same as an increased risk of having new disease (incidence). A more accurate HR for age could be determined by the rate of progression or the risk of morbidity. In other words, is the progression of MMVD to CHF the same for dogs of different age, and the risk of MMVD the same in all age groups? These questions warrant further prospective studies, where proper statistical considerations should be made in advance. The significant age-related increase in NTproANP and NOx previously described in healthy dogs15 was not detected in this study of diseased dogs. This is, however, not surprising, since the study was lacking healthy controls and all dogs were middle-aged or older. Consequently, there were no age-matched controls.

Endothelial function and nitric oxide Our finding of inverse impact on HR by NOx concentrations in Cox models (Table 4) is partly in contrast to a previously reported decreased NOx concentration in untreated MMVD dogs.29 However, the designs of these two studies are different. Our study was not designed to explore changes in NOx by degree of MMVD. On the other hand, decreased NOx concentrations found in the previous study may have been confounded by age-stratified changes in NOx.15 An alternative explanation of this incongruence is related to the complex signaling relationship and feedback mechanisms between NOx, natriuretic peptides, and other mediators. Specifically, endothelial dysfunction decreases local capability of cell NO production. On the other hand, with maintained endothelial function, NO in combination with the natriuretic peptides is an important beneficial counter-regulatory factor.43 Therefore, the decreased hazard with increase in initial NOx concentrations could indicate that dogs in our study initially had normal endothelial function and could increase their endothelial NO production to maintain peripheral blood flow. This explanation seems plausible, because mean age at enrollment was 6.6  2.1 years, whereas arteriosclerotic changes reported in necropsy material are from older animals (median 11 years).25 On the other hand, by use of indirect measures (flow measurements) peripheral endothelial dysfunction

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Figure 3 Panel A. Regression lines for NT-proANP for the 36 months before heart failure or censoring in 78 dogs with mitral regurgitation. The dashed line describes the time course of NT-proANP in dogs developing congestive heart failure (CHF) and the solid line dogs not reaching CHF before end of follow-up (censored). Panel B. The corresponding true trajectories of NT-proANP values for dogs not reaching CHF during the surveillance period (censored). Panel C. The corresponding true trajectories of dogs for dogs reaching CHF during the surveillance period. All regression lines are smoothed with the spline technique.

has been reported to decrease by degree of MR.27 Consistent with this, we found that dogs reaching CHF had lower NOx than censored dogs. This fits to the theory that compensatory and responsive increase in NO production is physiological and beneficial, whereas decreased responsiveness, i.e. endothelial dysfunction, is an important cardiovascular risk factor.43,44 Unfortunately plasma NOx levels are influenced by gastrointestinal sources (digested protein, bacterial source, vegetable NOx content, etc.). Although blood samples were taken from fasted animals, we cannot exclude the possibility that some animals could have ingested food, thus contributing to the high variability in plasma NOx concentrations compared to NTproANP. Taken together, the high variability in plasma NOx limit the value of these measurements in the clinical setting. More specific biomarkers of endothelial function and further longitudinal studies with age-matched controls are needed to verify the extent and time course of endothelial dysfunction in dogs with MMVD.

Heart murmur An increase in the grade of murmur (3/6) and an increased heart rate (130/min) were identified as risk factors in univariable analyses (Tables 2 and 4). These findings are in concordance with earlier reports.30,42,45 Although both predictors were significant as baseline variables in the Cox model, the baseline analysis is biased because one study inclusion criterion was an audible heart murmur; thus, we know that these animals are at risk before inclusion.2 However, the results of the longitudinal Cox models were also significant for both predictors. Thus, an increase in HR was found when

the grade of murmur increased from intermediate (grade 3e4, HR ¼ 9.5) to loud (grade 5e6, HR ¼ 17), compared to a baseline HR of only 2.1 (Tables 2 and 4, respectively).

Heart rate Increased heart rate has earlier been identified to have diagnostic value in identifying dogs in CHF.3,46 In a meta-analysis of 256 dogs with MMVD,42 heart rate was not among the variables associated with a progression of heart failure class or survival. In cross-sectional studies, heart rate variability47 and mean heart rate48 changed with progression of MR, but heart rate itself and increase in heart rate, as a predictors of CHF, have to date not been evaluated in longitudinal models (Fig. 2, Panel B and Table 4). The weak significance of heart rate in the univariable baseline analysis (HR ¼ 1.21, p ¼ 0.03) is in agreement with the previous meta-analysis.42 Some dogs entering our study had a low-grade murmur with no heart enlargement and the heart rate of these dogs was not expected to be increased,48 as reflected in a low baseline hazard of all dogs included. On the other hand, in the longitudinal model, which analyzed the change in absolute heart rate for every individual, and specifically when this HR was split into intervals, a heart rate >130 was identified as a significant risk (Fig. 2, Panel D and Table 4). This is in agreement with a previous report describing correlations between heart, lifespan and cardiovascular mortality risk in humans as well as in other mammals.49 Taken together, these results suggest that there is a true increase in risk of CHF both for increase in degree of murmur and heart rate.

NT-proANP predicts risk of congestive heart failure in mitral regurgitation

Limitations Only dogs of one breed (CKCS) were recruited. This is a limitation in that results may not be directly applicable to other breeds. However, although CKCS have a high prevalence of MMVD, the breed has not been shown to differ in how the disease is expressed. The survival after CHF for CKCS in the QUEST study was equal to or longer than for other breeds50 and the possibility to exclude potential confounding variables (e.g., between breed variation) in this study is considered an advantage. The small sample size and high variability of NOx measures decreased the power of the analysis to identify all aspects of endothelial function in MMVD. This was a clear limitation, specifically because both natriuretic peptides and NO exert their action via cGMP. A few dogs had single missing NT-proANP measurements in the data set (N ¼ 24 out of 312 visits). Although we could not analyze whether missing values were missing by chance, the effect of missing values on longitudinal models was considered minimal, because the number of missing values was small and most missing NT-proANP measurements (N ¼ 7) were at the final visit, not used in the prediction models. However, although the extended Cox model used was robust, missing values may have a marginal effect on results. Finally, in addition to other signs, CHF could not be confirmed without a diagnostic thoracic radiograph. In a previous report, the agreement between and within readers with different experience was only moderate.51 Therefore some dogs may potentially have been classified as erroneously having/not having pulmonary edema or congestion. However, all researchers were experienced readers and all radiographs were reviewed by consensus at annual meetings, using previous radiographs for comparison. Therefore within and between researcher variability for judging initial onset of pulmonary edema was most likely minimal. Nevertheless, mild pulmonary edema is a subtle sign on which exposure factors, film type and contrast may have an impact.52

Conclusions This long-term study demonstrates that increased levels of blood NT-proANP, particularly levels above 1000 pmol/l, as well as an increase in the degree of heart murmur (grade 3/6 and above) and heart rate (above 130 beats/min) are associated with an increase in hazard of CHF in CKCS dogs with MMVD. The estimated time to CHF for dogs

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with NT-proANP levels above 1000 pmol/l was 9e14 months. Single and follow-up measurements of blood NT-proANP may add to both diagnostic and prognostic accuracy of dogs in the late course of MMVD. Endothelial dysfunction, measured as plasma NOx, is likely a contributing factor to heart failure. As a result, further studies using appropriate longitudinal statistical analyses to compare the beneficial profile of both NT-proANP and NTproBNP as well as measures of endothelial function in different clinical settings are warranted.

Conflict of interest The authors declare no conflicts of interest.

Acknowledgments We thank Dr. Peter Lord and Dr. Gretchen Repasky for kindly reviewing the language.

Appendix Increased NT-proANP predicts risk of congestive heart failure in Cavalier King Charles spaniels with mitral regurgitation caused by myxomatous valve disease. Anders S. Eriksson, DVM, PhD, Jens Ha ¨ggstro ¨m, DVM, PhD, Henrik Duelund Pedersen, DVM, DrVetSci, Kerstin Hansson, DVM, PhD, AnnaKaisa Ja ¨rvinen, DVM, PhD, Jari Haukka, PhD, Clarence Kvart, DVM, PhD.

Statistical methods Cox regression models Consecutive measurements for dogs were not independent, meaning that the same dog contributed n times to the data set. The dependence was accounted for by use of a cluster- term (“dog”) in the model formula, indicating there were multiple observations (clusters) from the same subject and requiring robust standard errors be produced for the coefficient estimates. Robust standard errors are designed to account for the nonindependence of observations from the same subject.40 Furthermore, after the variation of the HR by level of continuous variables had been evaluated using penalized smoothing

152 splines (Fig. 2), we analyzed interval HRs by cutting the value of a predictor into intervals. The interval HR expresses the HR for the respective interval compared to a reference interval. We coded continuous baseline variables as constants. This “baseline constant” was evaluated from beginning to end, thereby benefitting all time-points. This is analogous to a predictor, which does not change during the study, such as gender. In the longitudinal time model, predictors vary as measured, and the model evaluates change in all time periods. For description of terms and a reference manual, please refer to link: http://cran.rproject.org/web/packages/survival/index. html.

Graphical prediction of NT-proANP trend Backward trends of NT-proANP from time of heart failure are presented in Figure 3. However, since dogs entered the study with different grade of MR, visualization of real forward trends are difficult to present. We therefore constructed a graphical simulation on how future individual NT-proANP trend over time (after inclusion in the study, i.e. time zero) by utilizing the mixed effect model smoothed by the splines method (Fig. A). By use of low degrees of freedom the model shows smoothed long term increase in NTproANP.

Figure A Prospective time course of N-terminal pro A-type natriuretic peptide (NT-proANP) blood concentrations showing all dogs with mitral regurgitation from inclusion in study. Note that the time scale is different from Figure 3; here drawn from where dogs enter the study in different disease stage and therefore present individual times in study. The gray lines show genuine individual trajectories of NT-proANP and red lines estimated increase in NT-proANP per day from baseline level

A.S. Eriksson et al. (i.e. inclusion day). In other words, red lines describe the estimated (smoothed) progression of increase in plasma NT-proANP by time from baseline level when dogs enter the study. The graphical model (red lines) is derived from an extended Cox’s model, based on a mixed effect model with time as fixed covariate and subject as random component. The effect of time is smoothed using the spline technique (degrees of freedom ¼ 3).

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