Atrial natriuretic peptide and cardiac troponin I concentrations in healthy Warmblood horses and in Warmblood horses with mitral regurgitation at rest and after exercise

Journal of Veterinary Cardiology (2013) 15, 105e121

www.elsevier.com/locate/jvc

Atrial natriuretic peptide and cardiac troponin I concentrations in healthy Warmblood horses and in Warmblood horses with mitral regurgitation at rest and after exercise* Dagmar S. Trachsel, Dr. med. vet. a,b,*, Colin C. Schwarzwald, Dr. med. vet., PhD a,c,d, Caroline Bitschnau, Dr. med. vet. a, Beat Grenacher e, Michael A. Weishaupt, Dr. med.vet., PhD a a

Equine Department, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 260, 8057 Zurich, Switzerland b Graduate School for Cellular and Biomedical Sciences, University of Bern, Freiestrasse 1, 3012 Bern, Switzerland c Center for Integrative Human Physiology (ZIHP), University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland d Center for Applied Biotechnology and Molecular Medicine (CABMM), University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland e Institute of Veterinary Physiology, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 260, 8057 Zurich, Switzerland Received 3 July 2012; received in revised form 30 November 2012; accepted 30 December 2012

KEYWORDS Equine; Cardiac biomarkers; Exercise capacity; Pulmonary wedge pressure

Abstract Objective: Atrial natriuretic peptide (ANP) and cardiac troponin I (cTnI) serve as biomarkers for increased cardiac pressure/volume loading and for myocardial stress or damage. The objective was to describe the time course of plasma ANP concentrations (CpANP) and plasma cTnI concentrations (CpcTnI) in horses with mitral regurgitation (MR) compared to healthy horses at rest and after exercise, and to describe the relationship of CpANP with cardiac dimensions and intracardiac pressures. Animals: 15 healthy Warmblood horses and 7 Warmblood horses with MR. Methods: Cardiac dimensions at rest were measured using echocardiography. All horses underwent standardized treadmill exercise. Biomarker concentrations and

*

Parts of the study have been presented at the ECEIM Congress in Edinburgh, Scotland, February 2e4, 2012. * Corresponding author. E-mail address: [email protected] (D.S. Trachsel).

1760-2734/$ - see front matter ª 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jvc.2012.12.003

106

D.S. Trachsel et al. intracardiac pressures were measured at rest and after exercise. Hypotheses were tested using statistical methods. The level of significance was P < 0.05. Results: Horses with MR showed increased left atrial (LA) and left ventricular (LV) dimensions but similar exercise capacity compared to healthy horses. Pulmonary capillary wedge pressures (PCWP) and CpANP increased with exercise. Horses with MR had higher PCWP and higher CpANP at rest and after exercise compared to healthy horses, with the maximum difference in CpANP reached 10 min after exercise. CpANP was significantly related to PCWP and e although inconsistently and only in healthy horses e to echocardiographic indices of LA and LV size and function. CpcTnI was low throughout the study in both groups. Conclusions: CpANP is increased in horses with MR and is related to LA pressures and to left heart dimensions. MR is not necessarily associated with exercise intolerance and exercise-induced myocardial stress or damage. ª 2013 Elsevier B.V. All rights reserved.

List of abbreviations: 2DE two-dimensional echocardiography Active LA FAC active left atrial fractional area change Active:total LA AC active-to-total left atrial area change ANP atrial natriuretic peptide AoD aortic diameter BWT body weight CO cardiac output plasma ANP concentration CpANP CpcTnI plasma cTnI concentration cTnI cardiac troponin I CV coefficient of variation d end-diastolic ECG electrocardiography ELISA enzyme linked immunosorbent assay HR heart rate heart rate reached when lactate concentration is 2 mmol/L HR2 HR4 heart rate reached when lactate concentration is 4 mmol/L HRR heart rate recovery interventricular septal thickness at end-diastole IVSd IVSs interventricular septal thickness at peak systole LA left atrium left atrial area prior to active contraction, at the onset of the electrocardiographic P wave LAAa maximum left atrial area at end-systole, prior to mitral valve opening LAAmax LAAmax normalized to a BWT of 500 kg LAAmax [500] LAAmim minimum left atrial area at end-diastole, after mitral valve closure maximum left atrial diameter at end-systole, prior to mitral valve opening LADmax maximum left atrial short-axis area at end-systole LAsxAmax LA RI left atrial reservoir index LV left ventricle LV EF left ventricular ejection fraction LV FAC left ventricular fractional area change LV FS left ventricular fractional shortening left ventricular internal area at end-diastole LVIAd LVIAd normalized to a BWT of 500 kg LVIAd [500] left ventricular internal area at peak systole LVIAs left ventricular internal diameter at end-diastole LVIDd

Cardiac biomarkers in horses LVIDs LVIVd LVIVs LVFWd LVFWs MR MWT PAD PAH PAP PAPm Passive LA FAC PCWP RIA ROC RWT s SV V2 V4 V150 V200

107

left ventricular internal diameter at peak systole left ventricular internal volume at end-diastole left ventricular internal volume at peak systole left ventricular free wall thickness at end-diastole left ventricular free wall thickness at peak systole mitral regurgitation mean wall thickness pulmonary artery diameter pulmonary arterial hypertension pulmonary artery pressure mean PAP passive left atrial fractional area change pulmonary capillary wedge pressure radio immunoassay receiver operator characteristics relative wall thickness peak systolic stroke volume speed reached at a lactate concentration of 2 mmol/L speed reached at a lactate concentration of 4 mmol/L speed reached at a heart rate of 150 beats/min speed reached at a heart rate of 200 beats/min

Introduction Exercise intolerance is a frequent clinical concern encountered in all equine sport disciplines. The most common causes of exercise intolerance include diseases of the musculoskeletal system, respiratory diseases, and anomalies in the cardiovascular system.1e4 Mitral regurgitation (MR) is one of the cardiac diseases that can impair exercise capacity in athletic horses.5,6 It causes left atrial volume overload and stretch of atrial myocytes, leading to left atrial (LA) dilation, progressive loss of atrial mechanical function, and increased risk for atrial arrhythmias.7 Despite the great compensatory reserve of the equine cardiovascular system, the impact of impaired atrial function might become apparent during high intensity exercise in horses with MR.8 The prevalence of heart murmurs and rhythm disturbances is high, even in apparently healthy sports horses.9e15 Despite some recent advances in cardiovascular diagnostics, the assessment of the contribution of cardiovascular diseases to loss of performance remains difficult. In clinical practice, diagnosis relies mainly on physical examination at rest and on electrocardiography (ECG) at rest and during exercise. Stress echocardiography has been used at some institutions to characterize cardiac mechanical function after exercise. However, methods are not standardized and the clinical value of stress echocardiography in horses is still unresolved.16 Other examination techniques such

as intracardiac pressure recordings are invasive and difficult to perform during or after exercise. Measurement of pulmonary capillary wedge pressure (PCWP), a surrogate of left atrial pressure,17e19 has been described in resting horses20,21 as well as in horses exercising on a treadmill.22e27 Both, healthy horses22e26 and horses with MR27 show an increase in PCWP during exercise compared to baseline. Furthermore, horses with moderate MR tend to have higher PCWP at rest and during exercise compared to healthy horses,27 reflecting haemodynamic consequences of valvular regurgitation. In the future, cardiac biomarkers might gain importance in the diagnostic workup of horses with exercise intolerance. Candidate biomarkers include natriuretic peptides (as markers for increased haemodynamic load)28,29 and cardiac troponins (as markers for myocardial stress or injury).28,30 Atrial natriuretic peptide (ANP) is a hormone that is physiologically secreted by atrial myocytes, but may also be expressed in diseased ventricular myocytes.31 It is a functional antagonist of the renin-angiotensinaldosterone system that is secreted in response to myocardial stretch due to pressure or volume overload.31,32 Storage of ANP in the equine atrial myocytes has been demonstrated.33,34 Unlike other natriuretic peptides (e.g., B-type natriuretic peptide), the amino acid sequence of the biologically active part of equine ANP shows 100% homology with human ANP.35 This allows for the use of commercially available assays for measurement of equine ANP.

108 Results from previous studies indicated that plasma ANP concentration (CpANP) can be increased in horses with altered LA and/or LV dimensions.36,37 Other studies in healthy horses showed an exerciseinduced increase in CpANP, which was considered a physiological response to increased heart rate (HR), increased atrial pressure, and/or changes in blood volume.38e44 Cardiac troponin I (cTnI) is a constitutive protein of the contractile apparatus of the cardiac muscle.45 Increased blood cTnI concentrations are used for diagnosis of irreversible myocardial injury in humans28,30 and have been documented in horses with severe myocardial lesions.46e49 The concept of release of the free cytosolic cTnI pool after cell membrane leakage due to mild, reversible injury has also been described.50,51 Variable increases in cardiac troponin concentrations have further been described after high intensity exercise in apparently healthy people,52e54 dogs,55 and horses,56e58 likely resulting from transient myocardial membrane injuries. In horses, the peak of a mild rise in plasma cTnI concentration (CpcTnI) was observed between 3 and 6 h after the end of exercise.57,58 The goal of this study was to describe the time course of CpANP and CpcTnI, respectively, at rest and immediately after exercise, in Warmblood horses with and without MR, and to relate CpANP to chamber dimensions, chamber function, and LA pressures. We hypothesized that CpANP can be used to differentiate horses with MR from healthy horses at rest and after a standardized exercise test. Furthermore, we hypothesized that exerciseinduced increases in cTnI are more pronounced in horses with MR compared to healthy horses, indicating increased myocardial stress.

Animals, material and methods Animals The horses were actively recruited for the study. Selection criteria included Warmblood breed, age between 6 and 15 years, and use as a performance horse (dressage and/or show jumping). Horses of both groups were used at a similar level of training or competition. The soundness of healthy horses (Group H ) was assessed by medical history, a thorough clinical examination, and routine haematology and plasma biochemistry analyses (CCS, DST). Musculoskeletal disease was excluded by an orthopedic examination including walking and trotting the horse on firm ground (MAW, DST, CB). Respiratory disease was excluded by auscultation, airway endoscopy, and tracheal wash cytology

D.S. Trachsel et al. (CCS, MAW, DST). Cardiovascular disease was excluded by cardiac auscultation, ECG, and echocardiography at rest (CCS, DST). The horses with MR (Group MR) had to fulfil the same criteria with the exception that they suffered from MR. Suspected exercise intolerance, as reported by the owners, was not an obligatory inclusion criteria for MR horses. Horses with structural cardiac disease other than MR, pathologic cardiac arrhythmias (including atrial fibrillation), or signs of congestive heart failure were not included. The study was approved by the district veterinary office of the canton Zurich. Owners’ consent was obtained prior to inclusion in the study. Fifteen healthy Warmblood horses (age 10
4 years [mean
SD]; body weight (BWT) 589
47 kg; height 170
4 cm) and 7 Warmblood horses with MR (age 12
5 years; BWT 595
26 kg; height 169
4 cm) were included in the study. The two groups were well matched for age (P ¼ 0.49; Student’s t-test), BWT (P ¼ 0.73), and height (P ¼ 0.54). All horses were used at the intermediate level for show jumping and dressage, except one in the Group MR which was only used for show jumping. Among the horses with MR, none had a history of exercise intolerance. All horses with MR had a grade 3-6/6 left-sided heart murmur. The two study populations were extensively characterized by transthoracic echocardiography, standardized exercise testing, and intracardiac pressure measurements, since the results of this study need to be interpreted with respect to the severity of MR and cannot be extrapolated to different populations undergoing different type of exercise.

Echocardiographic measurements Standardized transthoracic two-dimensional (2DE), M-mode, and Doppler echocardiography was performed during the first examination.59,60 Echocardiographic recordingsf were obtained by an experienced clinician (CCS) and all measurements were performed offline from the digitally stored cine loop recordings by the same examiner. Three non-consecutive cycles were measured and averaged for all echocardiographic variables. The echocardiographic examination included measurements of the aortic (sinus) diameter (AoD) and the maximal pulmonary artery diameter (PAD) obtained from right parasternal long-axis 2DE recordings of the left and right ventricular outflow tract, respectively, at end-diastole. Left ventricular (LV) dimensions and LV systolic function, as f GE Vivid 7 Dimension (BTO6) with a M4S Phased Array Transducer, GE Healthcare, Milwaukee, WI, USA.

Cardiac biomarkers in horses well as LA dimensions and LA mechanical function were assessed using previously described imaging standards.16,59e62 Linear echocardiographic indices of LA and LV size included: Maximum LA diameter measured in a right parasternal long-axis 2DE recording at endsystole (LADmax); and interventricular septal thickness (IVSd, IVSs), left ventricular internal diameter (LVIDd, LVIDs), and LV free wall thickness (LVFWd, LVFWs) measured in a right parasternal short-axis Mmode recording at the level of the chordae tendineae at end-diastole (d) and peak systole (s); relative wall thickness at end-diastole [RWT ¼ (IVSd þ LVFWd)/LVIDd]; and mean wall thickness at end-diastole [MWT¼ (IVSd þ LVFWd)/2]). The LV fractional shortening (LV FS) was calculated as an uni-dimensional index of LV systolic function [LV FS ¼ (LVIDd e LVIDs)/LVIDd  100]. Uni-dimensional (linear) indices of LA function are not considered reliable61 and therefore were not calculated. Two-dimensional (area-based) indices of LA and LV size included: Maximum LA area at end-systole prior to mitral valve opening (LAAmax), LA area at the onset of the electrocardiographic P wave prior to active contraction (LAAa), and minimum LA area at the time of mitral valve closure (LAAmin), all measured in a right parasternal long-axis 2DE recording; maximum LA area measured in a right parasternal short-axis 2DE recording at end-systole (LAsxAmax); and LV internal area measured in a right parasternal long-axis 2DE recording at enddiastole (LVIAd) and at peak systole (LVIAs). Two-dimensional (area-based) indices of LA and LV function included: Active LA fractional area change [Active LA FAC ¼ (LAAa e LAAmin)/ LAAa  100]; passive LA fractional area change [Passive LA FAC ¼ (LAAmax e LAAa)/LAAmax  100]; active-to-total LA area change [active:total LA AC ¼ (LAAa e LAAmin)/(LAAmax e LAAmin)]; LA areabased reservoir index [LA RI ¼ (LAAmax e LAAmin)/ LAAmin]; and LV fractional area change [LV FAC¼ (LVIAd e LVIAs)/LVIAd  100]. Three-dimensional indices of LV function included: Ejection fraction [LV EF ¼ (LVIVd e LVIVs)/ LVIVd  100], stroke volume [SV ¼ LVIVd e LVIVs] and cardiac output [CO ¼ SV  HR], where LV internal volume at end-diastole (LVIVd) and at peak systole (LVIVs) were calculated by the single-plane Simpson’s method based on the right parasternal long-axis 2DE recordings.16,63 The measurements of chamber dimensions were corrected for differences in BWT according to the principles of allometric scaling.64,65 Specifically, the measurements of chamber dimensions were normalized to a BWT of 500 kg [500] using the following

109 equations: Chamber diameter [500] ¼ Measured chamber diameter/BWT1/3  5001/3; and chamber area [500] ¼ Measured chamber area/ BWT2/3  5002/3. Grading of valvular regurgitations was based on a subjective grading scale (1/9 to 9/9) considering the high-velocity jet area, the regurgitant signal duration, and the number of imaging planes in which the high-velocity jet could be observed in the receiving chamber.13

Exercise testing All horses were accustomed to the treadmillg over 2 days with at least 3 training sessions lasting 40 min each. On the day of measurements, the horses were instrumented for measurements of HR by telemetric ECGh and for intracardiac pressure measurements (see below). A catheteri was placed into the left jugular vein for blood collection. The horses were then warmed-up for approximately 30 min at all 3 gaits. Subsequently, they underwent a submaximal incremental exercise test on the treadmill inclined at 6%. The time between beginning of the warm-up and the exercise test was 54
11 min. The exercise test consisted of 2 stages at trot (3.5e4.0 m/s) and 2 (Group H: n ¼ 0; Group MR: n ¼ 1), 3 (Group H: n ¼ 8; Group MR: n ¼ 4) or 4 (Group H: n ¼ 7; Group MR: n ¼ 2) stages at canter and gallop (6.0e10.0 m/s). Depending on the horse’s individual gait performance, the trot-to-canter transition was at 6.0 or 6.5 m/s. Speed increments were 1 m/s. The first stage lasted 2 min, all subsequent stages lasted 90 s. The horses were not run to exhaustion; instead, the exercise test was terminated when the horses reached a speed at which the HR was >190/min and the blood lactate concentration was >4 mmol/L (aerobic-anaerobic threshold). The time from peak exercise to complete stop of the treadmill was 30 s. Then the treadmill was stopped for 5 min of passive recovery before the horses walked for active cool-down for 30 min. HR was registered continuously and blood lactate concentrationsj were measured before warm-up, at the end of every step of the exercise test, and at 30, 60, 120, 180 and 300 s after completion of exercise. To objectify the variation in exercise capacity between the study horses, the following indices were calculated: Speed reached at an HR of 150 and 200 beats/min (V150 and V200); speed reached at a blood lactate concentration of 2 and 4 mmol/L (V2 and V4); and HR reached when lactate g h i j

Mustang 2200, Graber AG, Fahrwangen, Switzerland. Televet 100, KruuseA/S, Marslev, Denmark. Intranule, 13G, Vygon, Ecouen, France. Lactat Pro, Axon Lab AG, Baden-Da ¨ttwil, Switzerland.

110 concentration was 2 and 4 mmol/L (HR2 and HR4). Heart rate recovery (HRR) was assessed during the first 5 min after the end of the exercise test by calculating the % of maximal HR at every 5 s interval.66

Intracardiac pressure measurements For intracardiac pressure measurements, a 7F double lumen Swan-Ganz catheterk of 110 cm length was introduced into the right jugular vein through an introducer sheathl and advanced into the pulmonary artery. Pulmonary artery pressure (PAP) and PCWP were measured using a fluid-filled system. The reference point was the point of the shoulder. The transducerm was connected to the catheter with a tubingn of 2 m length. Pressure data were displayed and digitally stored on a physiologic monitor system.o Baseline recordings of intracardiac pressures at rest were started when the horse stood quiet on the treadmill. Further recordings were obtained after warm-up and before starting the exercise test, immediately at the end of the exercise test (0 min), and 5, 15, and 30 min after the end of the exercise test. PCWP was measured by inflating the balloon of the Swan Ganz catheter until wedging was confirmed on the display of the monitor. At each time point, up to four PCWP recordings were obtained, measured, and averaged for further analyses. When, during a 3-min period, accurate PCWP recordings could not be obtained or the balloon could not be wedged despite repositioning of the catheter, the time point was marked as missing and the study was continued in order to stay on the time schedule. Mean pulmonary artery pressures (PAPm) were measured offline based on the digital data files, using dedicated analysis softwarep. At each time point, 5 consecutive beats were measured and averaged for further analyses.

Collection of blood samples Blood samples for baseline CpANP and CpcTnI, respectively, were collected the day before the exercise test in a quiet environment through puncture of the jugular vein. The day of the exercise k

Edwards lifesciences, Horw, Switzerland. Intro-flex 8,5F, Edwards liefsciences, Horw, Switzerland. m DTXplus, Becton Dickinson, Allschwil, Switzerland. n Pressure tubing 80IN, 200 cm, Becton Dickinson, Allschwil, Switzerland. o Cardiocap, Anandic medical systems AG, Diessenhofen, Switzerland. p S/5 Collect software, Datex Ohmeda Division, Finland. l

D.S. Trachsel et al. test, blood samples were collected through the intravenous catheter placed in the left jugular vein. Samples for measurement of CpANP were collected at the following time points: before starting the exercise test (after warm-up), immediately at the end of the exercise test (0 min), and at 5, 10, 15, 30, 60, 120, 180, 240, 360 min, 10 h, and 24 h after exercise. Samples for measurement of CpcTnI were collected at the following time points: before starting the exercise test (after warm-up) and at 5, 60, 120, 180, 240, 360 min, 10 h, and 24 h after exercise. Plasma samples for measurement of CpANP were harvested into chilled tubes containing potassium-EDTAq and aprotininr and cooled on ice (4  C). Within 15 min after collection, the tubes were centrifuged for 5 min at 4  C and at 1570 g. The plasma was then harvested, transferred into cryovialss, and frozen at 80  C. For cTnI measurements, plasma was collected into lithium heparin tubes and further processed as described for the ANP samples. All samples were stored at 80  C for the duration of 7e26 months until analysis. Stability for ANP (at least 12 months at 80  C)67 and for cTnI (up to 3 years at 70  C)68 had previously been established by others, but was not specifically assessed within the scope of this study.

Laboratory analyses The CpANP was measured using a commercially available radio immunoassay (RIA) kitt according to the manufacturer’s instructions. For extraction, the samples were acidified with an equal amount of 1% trifluoroacetic acid (in water). Subsequently, they were centrifuged for 20 min at 16 100 g and at 4  C. The supernatants were applied onto Strata C18-E solid phase extraction columnsu (sorbent mass 200 mg/3 mL volume), conditioned with 60% acetonitril in 1% trifluoroacetic acid (in 39% water), and washed three times with 1% trifluoroacetic acid (in water). The columns were then washed twice with 1% trifluoroacetic acid (in water) before the elution was processed with a 60% acetonitrile solution in 1% trifluoroacetic acid (in 39% water). The eluent was then evaporated to dryness in a speed vac freeze dryerv. Before starting the RIA, the samples were reconstituted in adequate buffer furnished by the manufacturer q

1.6 mg EDTA/mL blood, Sarstedt AG, Sevelen, Switzerland. Lyophilized aprotinin (600 KIU/mL blood), SigmaeAldrich Chemie GmbH, Buchs, Switzerland. s TTP Techno Plastic Products AG, Trasadingen, Switzerland. t Test Kit 2011; Peninsula Laboratories, member of the BACHEM group, San Carlos CA, USA. u Phenomenex, Torrance, CA. v Evaporation centrifuge RC10.10 Jouan Therma, France. r

Cardiac biomarkers in horses and split into two aliquots for duplicate measurements. Then the RIA procedure was conducted following the manufacturer’s instructions. The CpANP were calculated by comparing the counts registered with a gamma-counterw to the formerly established standard curve ranging from 10 to 1280 pg/mL. Duplicate measurements were averaged to obtain the final results. The ANP RIA is a competitive immunoassay working with polyclonal antibodies presenting 100% cross reactivity to human, bovine, and porcine ANP (1e28), to mouse, rabbit, and rat ANP(1e28), to rat ANP(8e33), to rat Atriopeptin III, to human antiparallel dimer for ANP(1e28), to Thr-Ala-Pro-Arg-ANP(1e28), to rat ANP(126e149), to human ANP(4e28) and to human ANP(7e28). This RIA had been validated and used in previous studies in horses.38,40 Our own data indicated that the intra-assay coefficient of variation (CV) was between 6 and 12% for concentrations between 12 pg/mL and 200 pg/mL, and 32% for low concentrations (<12 pg/mL). The inter-assay CV ranged between 21% and 60% for all tested concentrations. Dilution parallelism showed good linearity for all tested concentrations (R2 > 0.97)x. The cTnI was measured on a single aliquot with a commercially available two-side enzyme linked immunosorbent assayy (ELISA) following the manufacturer’s instructions. Formerly published validation data48 and data from our own laboratory showed an acceptable analytical performance (inter-assay CV  10%, intra-assay CV  10% for concentrations >0.28 ng/mL, increasing CV for decreasing concentrations, and CV  34% for concentrations  0.09 ng/L, dilution parallelism with good linearity (R2 > 0.97) for concentrations <2.5 ng/mLz. For human samples, the assay’s analytic sensitivity is 0.02 ng/mL and the range of reported concentrations is 0e50 ng/mL.

Data analysis and statistics Graphical presentation, data analyses, and statistics were performed using commercially

GAMMAmatic I, Kontron Analytical, W þ W Elecronic AG, Mu ¨nschenstein, Switzerland. x Analytic validation and comparison of three commercial immunoassays for atrial natriuretic peptide (ANP) measurement in plasma in horse. DS Trachsel, CC Schwarzwald, B Grenacher, MA Weishaupt, in preparation. y iSTAT, Axon Lab AG, Baden-Da ¨ttwil, Switzerland. z Analytic validation of a commercially available immunoassay (i-STAT) for plasma cardiac troponin I measurements in horses. DS Trachsel, MA Weishaupt, B Grencher, CC Schwarzwald, in preparation. w

111 available softwareaa,ab. Distribution and variance of the data were assessed by inspection of scatter plots, histograms, and normal probability plots. Normally distributed data were reported as mean
standard deviation (SD), non-parametric data were described as median (range). The level of significance was P ¼ 0.05. Population characteristics and echocardiographic measurements were compared between groups using Student’s t-test.aa For assessment of differences in the size of the great vessels, LA size, LA mechanical function, LV size, and LV systolic function, respectively, Bonferroni correction (i.e. critical P ¼ 0.05/number of comparisons) was applied to correct for multiple comparisons (see Table 1). The relationship between HR and velocity was assessed by linear regressionaa; the relationship between blood lactate concentrations and velocity and HR, respectively, were approximated by an exponential regressionaa; and HRR was described by a bi-exponential decay functionaa (Supplementary Table 1). Indices of exercise capacity (V150, V200, V2, V4, HR2, HR4) were extracted from these regression lines and compared between groups using Student’s t-test.aa Two-way repeated-measures (mixed model) ANOVAab was used to detect differences in intracardiac pressures over time (i.e. between baseline and post-exercise measurements) and between groups. When the F-test indicated significant differences, comparison of post-exercise measurements to resting values (baseline) was performed using a Holm-Sidak post-hoc test. Homogeneity of variances was assessed by graphical display of the data and validity of the normality assumption was confirmed by assessment of normal probability plots of the residuals. Data for CpANP were not normally distributed and displayed heterogeneous variance. Therefore, data were log transformed and analysed using a parametric two-way repeated-measures (mixed model) ANOVAab to detect differences over time (i.e. between baseline and post-exercise measurements) and between groups. When the F-test indicated significant differences, comparison to resting values was performed using a Holm-Sidak post-hoc test. Homogeneity of variances was assessed by graphical display of the data and validity of the normality assumption after transformation was confirmed by assessment of normal aa GraphPad Prism, version 5.03, GraphPad Software, San Diego, CA, USA, www.graphpad.com. ab SigmaStat, version 3.5, Systat Software GmbH, Erkrath, Germany.

112

D.S. Trachsel et al.

Table 1 Echocardiographic assessment of left atrial and left ventricular size and function in healthy horses and in horses with mitral regurgitation. Variables

BWT HR (during the echocardiography) Great vessels PAD AoD Left atrial size LADmax LAAmax LAsxAmax LADmax/LVIDd LAAmax/LVIAd Left atrial mechanical function Active LA FAC Passive LA FAC Active:total LA AC LA RI Left ventricular size IVSd LVIDd LVFWd IVSs LVIDs LVFWs RWT MWT LVIAd LVIAs Left ventricular systolic function LV FS LV FAC LV EF SV CO

Units

Group

P values (Student’s t-test)

Healthy (n ¼ 15) Mean
SD

MR (n ¼ 7) Mean
SD

kg min1

589
47 41
6

595
26 36
5

0.73 0.10

cm cm

6.9
0.5 8.0
0.6

6.9
0.3 8.1
0.6

0.95 0.67

cm cm2 cm2 e e

12.3 102.8 123.6 1.1 0.6








0.7 8.5 19.3 0.1 0.1

14.5 137.5 154.1 1.1 0.7








1.2 18.4 13.3 0.1 0.1

<0.0001 <0.0001 0.001 0.90 0.005

% % e %

18.1 24.9 0.3 64.1







6.5 4.9 0.1 11.9

13.6 23.7 0.3 54.0







10.5 5.5 0.2 19.3

0.22 0.61 0.32 0.14

cm cm cm cm cm cm e cm cm2 cm2

3.2 11.2 2.5 4.4 6.8 4.5 0.5 2.8 169.3 73.8













0.4 0.9 0.4 0.4 0.9 0.6 0.1 0.3 12.5 11.4

3.0 13.3 2.8 4.2 8.3 4.4 0.4 2.9 193.0 93.2













0.5 1.2 0.5 0.5 0.8 0.6 0.1 0.4 19.1 8.2

0.30 0.0002 0.69 0.44 0.0008 0.69 0.07 0.73 0.002 0.0006

40 56 72 929 40








5 5 4 113 10

37 46 68 1132 46








3 14 2 205 15

0.22 0.01 0.04 0.007 0.32

% % % mL mL/min

Significant after correction for multiple comparisons

(Critical P ¼ 0.025) n.s. n.s. (Critical P ¼ 0.01) * * * n.s. * (Critical P ¼ 0.0125) n.s. n.s. n.s. n.s. (Critical P ¼ 0.005) n.s. * n.s. n.s. * n.s. n.s. n.s. * * (Critical P ¼ 0.01) n.s. n.s. n.s. * n.s.

BWT, body weight; MR, mitral regurgitation; HR, heart rate; n.s.: not significant; *: significant for the indicated critical level. Measurements obtained from right parasternal long-axis 2DE recordings of the left atrium and the left ventricle16,61: AoD, Aortic sinus diameter at end-diastole; PAD, Pulmonary artery sinus diameter at end-diastole; LADmax, Maximum LA diameter at end-systole; LAAmax, Maximum LA area at end-systole; Active LA FAC, Active LA fractional area change; Passive LA FAC, Passive LA fractional area change; Active:total LA AC, Active-to-total LA area change; LA RI, area-based LA reservoir index; LVIAd, LV internal area at end-diastole; LVIAs, LV internal area at peak systole; LV FAC, LV fractional area change; LV EF, LV ejection fraction; SV, stroke volume; CO, cardiac output. Measurements obtained from right parasternal short-axis 2DE recordings at the level of the aorta and left atrium61: LAsxAmax, Maximum LA area at end-systole. Measurements obtained from right parasternal short-axis M-mode recordings of the left ventricle at the level of the chordae tendineae16,62: IVSd, Interventricular septal thickness at end-diastole; LVIDd, Left ventricular internal diameter at end-diastole; LVFWd, LV free wall thickness at end-diastole; IVSs, Interventricular septal thickness at peak systole; LVIDs, Left ventricular internal diameter at peak systole; LVFWs, LV free wall thickness at peak systole; RWT, relative wall thickness; MWT, mean wall thickness; LV FS, LV fractional shortening.

Cardiac biomarkers in horses probability plots of the residuals. Based on these analyses, the time points with the greatest differences in mean CpANP between groups were identified. For these time points, the differences in means (including their 95% confidence intervals) were calculatedaa based on the log-transformed data and then converted back to the original scale. Receiver operator characteristic (ROC) curvesaa were used to describe test characteristics of CpANP measurements and to identify the best cut-off concentration for distinguishing horses with MR from healthy horses at rest and after exercise. CpcTnI were expressed as median and ranges and no further statistical analyses were done, since most measurements were below the detection limit of the assay. Finally, the relationship between CpANP and PCWP and between CpANP and echocardiographic indices of cardiac size and function, respectively, was assessed. As colinearity was present between CpANP, PCWP, and several echocardiographic measurements (all being significantly larger in Group MR), Group H and Group MR were analysed separately. The relationship between PCWP and log (CpANP) was assessed by multiple linear regression analysisab including measurements obtained at baseline, after warm-up, and at 0, 5, 15, and 30 min after exercise. To account for repeated measures within subjects, horses were included in the model using dummy variables. Backwards stepwise multiple linear regression analysesab were performed to assess the relationship between weight-corrected echocardiographic indices of LA and LV size and function (LAAmax [500], LVIAd [500], Active LA FAC, and LV FAC) and log (CpANP) at baseline, log (CpANP) at 10 min post exercise (when the difference in CpANP was greatest between the groups), and highest log (CpANP) measured for each horse at any time point after exercise, respectively. Measurements of LA and LV dimensions were forced into the equation, whereas the indices of LA and LV systolic function were allowed to be eliminated by the regression model.

113 increased in Group MR, associated with an increased SV. The area-based ejection-phase indices of systolic LV function (i.e., LA FAC, LA EF) were lower in Group MR compared to Group H, but statistical significance was lost after correcting for multiple comparisons. The severity of the MR was graded as mild (grade 4/9 and 5/9) in 2 horses, mild to moderate (grade 6/9) in 3 horses, moderate to severe (grade 8/9) in 1 horse, and severe (grade 9/9) in 1 horse.

Exercise capacity All horses tolerated the exercise test well. No horse in Group MR showed any problems in reaching the fixed end-point of the exercise test (HR > 190/min and blood lactate > 4 mmol/L). The relationships between treadmill velocity, HR, and blood lactate concentrations, respectively, as well as HRR are shown in Figure 1. The values for V150 and V200 were 5.0
0.82 m/s and 8.8
0.98 m/s (mean
SD) in healthy horses and 5.6
0.23 m/s and 9.0
1.17 m/s for horses with MR, respectively. The values for V2 and V4 were 5.8
0.95 m/s and 7.0
0.99 m/s in healthy horses and 6.5
0.61 m/s and 7.6
0.36 m/s in horses with MR, respectively. Finally, HR2 and HR4 were 161
14 min1 and 176
15 min1 in healthy horses and 163
10 min1 and 180
11 min1 in horses with MR, respectively. None of the calculated performance indices differed significantly between groups (V150, P ¼ 0.09; V200, P ¼ 0.69; V2, P ¼ 0.08; V4, P ¼ 0.13; HR2, P ¼ 0.69; HR4, P ¼ 0.48). On the ECG recording, most of the horses showed a variable number of supraventricular or ventricular ectopic depolarizations at rest and during the recovery period. Quantitative analysis of these events and comparison between groups was not done, since the intracardiac catheter was expected to provoke a certain number of ectopic depolarizations and it was not possible to distinguish these ectopic depolarizations from exercise-induced ectopic depolarizations.

Intracardiac pressures

Results Echocardiography at rest Echocardiographic measurements are summarized in Table 1. Significant LA enlargement was evident in Group MR compared to Group H, whereas LA function was not significantly different between groups. Similarly, LV internal dimensions were

Pressure recordings could be obtained in 12/15 healthy horses and in 7/7 horses with MR at rest and during the recovery period immediately after the exercise test. Results are shown in Figure 2. Pulmonary artery pressure and PCWP after exercise were higher than resting values in all horses and decreased rapidly during the recovery period. PAPm was not significantly different between groups. However, horses with MR showed

114

D.S. Trachsel et al.

Figure 1 Characterization of exercise capacity in healthy horses (n ¼ 15, C) and horses with mitral regurgitation (n ¼ 7, ) during a submaximal incremental exercise test. AeC, Relationship between velocity, heart rate, and blood lactate concentrations. D, Heart rate recovery after exercise.

significantly higher PCWP than healthy horses at all time points (mean difference: 5 mmHg, P ¼ 0.001).

Biomarkers The CpANP are presented in Figure 3 and in Supplementary Table 2. In both groups, CpANP increased significantly after exercise, peaked between 0 and 10 min, and returned to baseline values within 120 min after exercise.

The CpANP were significantly higher in horses with MR compared to healthy horses at all time points (P ¼ 0.037). The largest differences between groups were observed at 5 min [CpANP in Group MR 2.5 fold higher (95% CI 1.3e4.8) than Group H] and 10 min [CpANP in Group MR 2.6 fold higher (95% CI 1.4e4.7) than Group H] after exercise. Further, the model showed that CpANP were significantly different from baseline until 60 min after exercise.

Figure 2 Mean pulmonary artery pressure (PAPm) and pulmonary capillary wedge pressure (PCWP) in healthy horses (n ¼ 12, C) and in horses with mitral regurgitation (n ¼ 7, ) before exercise (baseline), after warm-up, and at different time points after exercise. The horizontal lines represent the mean values. F-test results for PAPm: Difference between groups, P ¼ 0.30; difference over time, P < 0.001; interaction group  time, P ¼ 0.09. F-test results for PCWP: Difference between groups, P ¼ 0.001; difference over time, P < 0.001; interaction group  time, P ¼ 0.75. * represents significant difference to baseline (Holm-Sidak post-hoc test).

Cardiac biomarkers in horses

115

Figure 3 Plasma atrial natriuretic peptide concentrations (CpANP) in healthy horses (n ¼ 15, black) and horses with mitral regurgitation (n ¼ 7, red) before exercise (baseline), after warm-up, and at different time points after exercise. The line represents the median, boxes represent 25the75th percentiles, and whiskers represent minimum and maximum measurements. Results of F-test on log-transformed data: Difference between groups, P ¼ 0.037; difference over time, P < 0.001; interaction groups  time P ¼ 0.097. * represents significant difference to baseline (Holm-Sidak post-hoc test).

ROC analyses were performed for CpANP at rest and at 5 and 10 min after exercise (i.e. at the time of the largest differences in CpANP). The results are summarized in Table 2. At rest, the ANP assay was not useful to reliably detect MR (p ¼ 0.11). However, ROC analysis for measurements after exercise showed that test performance was best when CpANP was measured 10 min after exercise and when a cut-off of 332 ng/mL was used. CpcTnI were low throughout the study in both groups (Fig. 4 and Supplementary Table 2). For a large majority of measurements in both groups (i.e., 18-21/22 measurements per time point), the values were below 0.06 ng/mL, which is the proposed upper range for healthy horses.48 Furthermore, at each time point, 7-13/22 measurements were below the detection limit of the assay (0.02 ng/mL). No obvious difference was observed between groups, neither at rest nor after exercise. Regression analyses revealed that both in Group H and Group MR, log (CpANP) was significantly

Figure 4 Plasma cardiac troponin I concentrations (CpcTnI) in healthy horses (n ¼ 15, black) and horses with mitral regurgitation (n ¼ 7, red) before exercise (baseline), after warm-up, and at different time points after exercise. The solid lines represent the median, boxes represent 25the75th percentiles, and whiskers represent minimum and maximum measurements. The dotted horizontal line represents the upper range determined for healthy horses.48

related to PCWP (H: P < 0.001, R2 ¼ 0.510; MR: P < 0.001, R2 ¼ 0.648). The multiple regression analyses further showed that log (CpANP) at baseline in Group H could be predicted from active LA FAC (P ¼ 0.012), whereas LAAmax [500] (P ¼ 0.347) and LVIAd [500] (P ¼ 0.249) did not contribute significantly (power of analysis, 0.911); LV FAC was removed from the model during the stepwise procedure. At time point 10 min, log (CpANP) could be predicted from LAAmax [500] (P ¼ 0.019), whereas LVIAd [500] (P ¼ 0.225) did not contribute significantly (power of analysis, 0.730); LV FAC and active LA FAC were removed from the model during the stepwise procedure. Finally, the highest log (CpANP) measured after exercise could be predicted from LAAmax [500] (P ¼ 0.002), LVIAd [500] (P ¼ 0.011) and LV FAC (P ¼ 0.003) (power of analysis, 0.99); active LA FAC was removed from the model during the stepwise procedure. In Group MR, none of the echocardiographic measurements could predict log (CpANP) at any of the three time points, but power of the analysis was low because of the small

Table 2 Results of the ROC analyses, showing best cut-offs for plasma ANP concentration (CpANP) to identify horses with mitral regurgitation at rest and at 5 and 10 min after the end of the exercise test, respectively. Area under the curve [95% CI] P Best cut-off for CpANP (ng/mL) Specificity [95% CI] (%) Sensitivity [95% CI] (%)

Rest

5 min post exercise

10 min post exercise

0.71 [0.43e1.0] 0.11 97 100 [78.2e100] 57.1 [18.4e90.1]

0.83 [0.61e1.0] 0.01 455 93.3 [68.1e99.8] 71.4 [29.0e96.3]

0.83 [0.59e1.1] 0.01 332 93.3 [68.1e99.8] 85.7 [43.1e99.6]

116 dataset (baseline, 0.089; 10 min after exercise, 0.654; peak ANP after exercise, 0.541).

Discussion In this study, CpANP was increased in a population of horses with mild to severe MR presenting with significant left-sided chamber enlargement and increased PCWP but normal performance capacity. Both in healthy horses and in horses with MR, CpANP increased after exercise and returned to baseline values within 120 min. The difference in CpANP between healthy horses and horses with MR was largest at 5 and 10 min after exercise. Therefore, discrimination of both groups was best in the immediate post exercise period, indicating that CpANP measurements might be most useful in combination with an exercise test. The CpcTnI were generally low and were not significantly influenced by the level of exercise or the severity of the MR as it was present in our population. The study groups were characterized in detail by transthoracic echocardiography, standardized exercise testing, and intracardiac pressure measurements. Echocardiographic examination showed that the horses with MR had significant left-sided chamber enlargement in comparison to healthy controls. The LA measurements were in the upper range or above published reference ranges for Warmblood horses.60 However, LA mechanical function was not significantly affected by MR in this study group. The LV internal diameter was also larger in Group MR compared to Group H. However, it remained within the upper normal range for Warmblood horses,60 with the exception of one horse that had a markedly increased LVIDd. The LV SV was significantly increased in Group MR, indicating intact compensatory responses in relation to volume overload from MR. Due to LV enlargement in horses with MR compared to healthy horses, the increase in SV was achieved without an increase in LV FS, FAC, and EF, respectively, all of which even tended to be decreased in horses with MR. Notably, CO was not significantly different between horses with MR and healthy horses. All horses were able to perform a standardized submaximal incremental exercise test designed to reach the individual aerobic-anaerobic threshold without showing any obvious signs of exercise intolerance or poor performance. The established regression lines of HR in relation to velocity (Fig. 1 and Supplementary Table 1) in healthy horses and in horses with MR were similar to those found for other Warmblood horses in training66,69 and they

D.S. Trachsel et al. were close to those found in Thoroughbreds.70,71 Blood lactate measurements in relation to velocity and HR, respectively, showed a great individual variability with almost overlying curves between groups. Similarly, the HRR was almost identical in the two groups. Finally, none of the indices of exercise capacity was statistically different between the two groups and all of them were similar to the indices published for healthy Warmblood horses in training.66 Therefore, we conclude that the intensity of exercise was similar in both groups and that exercise capacity was not impaired in the examined population of horses with MR. However, these results are based on a relatively short treadmill exercise test and cannot be extrapolated to longer lasting, more exhaustive exercise. To further assess the haemodynamic consequences of the MR in our study population, intracardiac pressure measurements were obtained at rest and after exercise. The PAPm in healthy horses at rest was within the expected range.22e25,72,73 However, the mean resting PCWP was slightly lower than previously reported for healthy horses,21e27 likely because of differences in instrumentation and recording techniques. As expected from former studies,22e27,72,73 both PAP and PCWP increased after exercise in both groups, although increases were lower than anticipated. Since we used a fluid-filled catheter system, pressure recordings were limited to the periods when the horses were standing still on the treadmill before and immediately after exercise. This was in contrast to other studies, in which micromanometer-tipped catheters allowed pressure recordings during exercise.22e25,72,73 This could explain the lower PCWP and PAP measured after exercise in this study compared to other studies, since normalization of pressures in the postexercise period occurs rapidly72,73 and the timing of measurements was different between studies as a result of different recording techniques. Moreover, former studies, especially those examining Standardbreds and Thoroughbreds, used protocols with higher exercise intensity.22e25,72,73 In this study, there was no difference in PAPm between Group MR and Group H at any time point (Fig. 2), and none of the horses with MR showed pulmonary arterial hypertension (PAH) after the exercise test. In humans, absence of PAH has been proposed as a predictive sign of symptom free interval in degenerative MR.74 However, the PCWP was increased throughout all time points in horses with MR (Fig. 2). Together with echocardiographic LA enlargement, this finding indicated that the MR in our study population was hemodynamically

Cardiac biomarkers in horses relevant, even though exercise capacity was not reduced. Haemodynamic consequences of heart disease were further reflected by the increased CpANP in horses with MR. This finding was in agreement with a small number of former studies on horses with cardiac disorders.36,37,ac However, in our study population, ROC analyses revealed that CpANP measured at rest did not allow differentiating between horses with MR and healthy horses. Differences in CpANP between groups were larger in the immediate post-exercise period, indicating that haemodynamic consequences of MR are exacerbated by exercise. As a consequence, the larger difference in CpANP after exercise allowed better discrimination between diseased and non-diseased horses. Measurement of CpANP 10 min after exercise provided high specificity and moderate sensitivity at a cut-off concentration of 332 ng/mL. The wide confidence intervals for specificity and sensitivity were likely related to the small sample size. Therefore, these results will have to be confirmed in larger prospective studies. The mean difference in PCWP of 5 mmHg between groups was small. However, similar changes in atrial pressure measured in experimental settings75 and in clinical studies on dogs and cats76e79 were associated with significant increases in CpANP. Thus, the increased pressure load applied on the atrial wall in horses with MR appeared to be sufficient to induce ANP release from atrial myocytes and was likely to be responsible for at least part of the increase in CpANP. In fact, the results indicated that log (CpANP) was significantly related to PCWP. However, only approximately 50 and 65% of the variation in log (CpANP) could be explained by alterations in PCWP in Group H and Group MR, respectively. Therefore, other factors may play a role in triggering ANP release. The difference in CpANP between groups was larger after exercise, whereas the difference in PCWP remained constant over all time points. Heart rate and changes in blood volume may influence ANP release after exercise,38e44 but neither of these two factors explains the larger difference in CpANP after exercise in this study. Maximal HR was similar in both groups (Fig. 1) and changes in blood volume were unlikely to be different, since total plasma protein concentrations increased to a similar extent after exercise in both groups (data not shown). It is, however, possible ac Plasma ANP concentrations in horses with various heart diseases at rest. DS Trachsel, B Grenacher, CC Schwarzwald, abstract presented at the ACVIM forum, New Orleans (USA), June 2012.

117 that the myocardial response is not proportional to the pressure change, resulting in progressively larger ANP release as pressures rise. In healthy horses, there was an association of log (CpANP) at rest with active LA function, of log (CpANP) 10 min after exercise with LA size, and of maximum log (CpANP) after exercise with LA size as well as LV size and systolic function. However, the previously published association between CpANP and LA dimensions in horses with heart diseases37 could not be confirmed in this study. This might be explained by a more heterogeneous patient population in the former study or by the small number of examined horses in the present study, as suggested by the insufficient power of the analyses in this group. In summary, all these findings point at a complex regulation of ANP secretion in horses. In addition to atrial pressure and/or volume overload, other factors such as expression of ANP by remodeled LV myocytes, activation of the reninangiotensin-aldosterone system, and sympathetic stimulation could have contributed to the response of ANP secretion31,80,81 but were not assessed in the present study. Overall, the results of this study are in agreement with former studies on ANP in horses with cardiac disease.36,37,ac However, the population of the present study only included horses with MR, and the results should not be extrapolated to horses suffering from other heart diseases without further studies. Also, only a small number of horses fulfilled the inclusion criteria and could be recruited for the study. As a consequence, the power to detect a relationship between CpANP, cardiac dimensions, and intracardiac pressures, was limited by the small sample size, especially in horses with MR. The CpANP at rest and after exercise were considerably higher than values reported in other studies in healthy horses and horses with heart disease.36e44,82,83 Possibly, changes in test kit composition over time by the manufacturer could have changed the affinity of antibodies and thereby could have affected test results. Moreover, differences in sample processing might have influenced the results, since the test kit used in this study demands an extraction step that may largely influence the amount of peptide present in the eluted solution. Different extraction methods used in former studies might have contributed to the discrepancy in CpANP between studies. Further, the limited stability of the ANP molecule demands rapid separation and freezing of plasma. The use of aprotinin to reduce protein degradation is advised. In former studies, aprotinin was not always used and often there is poor description of

118 sample processing procedures. Hence, different sample collection and handling might have resulted in lower values in former studies. An important limitation of the present study is the suboptimal analytic performance of the ANP assay, in contradiction to previously published data, based on which the assay was originally chosen.38,40 The high test-retest variability might be related to the inherent imprecision of radio immunoassays, poor affinity of the antibodies for equine ANP, or poor adaptation of the test for horse plasma (matrix effect). Nonetheless, the analytic performance was overall similar to other immunoassays used in veterinary medicine,84e89 and only slightly lower than proposed in current guidelines for protein biomarkers.90 Despite the suboptimal analytic performance of the assay, significant differences in CpANP between groups could be detected on a population level. However, it is important to realize that the high variability and the poor analytic performance of the ANP assay limit its diagnostic validity for use in individual horses in a clinical setting. Furthermore, the results emphasize that comparisons of CpANP between groups or between individual horses require standardized sampling methods, sample processing, and analysis methods. In this study, CpcTnI was measured with the hypothesis that it would detect exercise-induced myocardial stress or damage. High cardiac troponin concentrations have been measured after intense exercise in human athletes.52e54 Similarly, CpcTnI have been shown to rise after high intensity exercise in horses.56e58 Highest values were seen during endurance competition in horses finishing in the top 10 and in eliminated horses after an 80 km race,56 indicating that cellular stress resulting in altered membrane permeability may occur in horses in the absence of obvious cardiac disease.56 In the same study, horses performing over 160 km had lower CpcTnI than horses competing at 80 km.56 A similar inverse relationship of cardiac troponin concentrations to duration of exercise has been shown in humans, and was explained by the higher training status of participants in longer events and exercise performed at lower intensity for longer distances.52 In our population completing comparably short exercise, CpcTnI measured at rest and after exercise were mostly below the detection limit of the assay; only a few samples reached higher post exercise values,56e58 and even in the presence of MR, the CpcTnI were not obviously raised after exercise. Despite the rapid elimination of cTnI in horses,91 the frequent sampling protocol used in our study would have allowed for the detection of the peak CpcTnI

D.S. Trachsel et al. expected 3e6 h after exercise.57,58 Therefore, we concluded that there was no obvious overload of the heart and absence of clinically relevant myocardial damage in horses with MR during short duration exercise to the anaerobic-aerobic threshold. However, no conclusions can be made in relation to potential myocardial damage in horses with more severe MR or after longer lasting, more exhaustive exercise.

Conclusion In conclusion, the results of this study indicate that CpANP is higher in horses with mild to severe MR that was characterized by increased left heart dimensions, increased PCWP, but normal exercise capacity and absence of clinical signs of congestive heart failure. The CpANP is partially related to PCWP (a surrogate of LA pressures) both in healthy horses and in horses with MR, indicating that ANP might serve as a marker of cardiac pressure loading. However, association with LA and LV dimensions and function was less consistent and in this study was only significant for a subset of echocardiographic indices in healthy horses. Although a similar relationship might be present in horses with MR, the small sample size in this study did not allow detecting or ruling out an association between CpANP and cardiac size and function in this group. Standardized treadmill exercise causes a disproportional increase in CpANP in horses with MR compared to healthy horses. Measurement of CpANP 10 min after exercise allows distinguishing horses with haemodynamic consequences of MR from healthy horses with a relatively high specificity and a moderate sensitivity, even when no reduction in exercise capacity is present. However, the diagnostic use of CpANP in individual horses in a clinical setting is limited by a suboptimal analytical performance of the ANP RIA. Finally, this study showed that MR is not necessarily associated with exerciseinduced myocardial stress or damage, since cTnI was low and was not influenced by the short exercise demanded in this population of horses.

Conflict of interest None.

Acknowledgements The study was founded by the Research Foundation of the University of Zurich, by the Foundation

Cardiac biomarkers in horses Forschung fu¨r das Pferd and the Na¨geli-Wolfermann Foundation. Further, we acknowledge Drs. K. Geser-von Peinen, N. Waldern and L. Ramseier collaborators of the Equine Performance Centre and the collaborators and students of the Clinic for Equine Internal Medicine of the Vetsuisse Faculty of the University of Zurich for their assistance.

Supplementary data Supplementary data related to this article can be found online at http://dx.doi.org/10. 1016/j.jvc.2012.12.003.

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