Corresponding author: Yasutomo Hori, Department of Small Animal Internal Medicine, Kitasato University, 23-35-1 Higashi, Towada, Aomori 034-8628, Japan; e-mail: email@example.com
Background: The clinical implications of evaluating C-terminal atrial natriuretic peptide (ANP) concentration in cats are still controversial.
Hypothesis: The objective of this study was to investigate the relationship between plasma C-terminal ANP concentration and left atrial pressure (LAP) in healthy cats with volume overload (study 1), and to compare plasma C-terminal ANP in normal cats and cats with cardiomyopathy (study 2).
Animals: Five healthy adult cats were used in study 1, and clinically healthy cats (n=8) and cats with cardiomyopathy (n=14) were used in study 2.
Methods: In study 1, cats were anesthetized and given acetated Ringer's solution (100 mL/kg/h for 60 minute) via the cephalic vein. Hemodynamic measurements and blood samples, collected from the jugular vein, were performed at 10-min intervals. In study 2, blood samples from normal cats and cats with cardiomyopathy were collected from the cephalic vein. The plasma C-terminal ANP concentration was determined by radioimmunoassay for human α-ANP.
Results: In study 1, volume overload significantly increased the C-terminal ANP concentration and LAP from baseline. The C-terminal ANP concentration was strongly correlated with the mean LAP. In study 2, age, E wave velocity, and the ratios of the left atrium to aorta were significantly higher in the cats with cardiomyopathy compared with the normal cats. The C-terminal ANP concentration was significantly higher in the cats with cardiomyopathy compared with the normal cats.
Conclusions and Clinical Importance: Our results suggest that the measurement of plasma C-terminal ANP in cats may provide additional information for the diagnosis of heart disease.
Recently, several hormones and peptides have been used as biomarkers for the diagnosis of heart disease, including natriuretic peptide, endothelin-1, and troponin I.1–4 C-terminal atrial natriuretic peptide (ANP) in cats consists of 28 amino acids, and shows physiological activity.5 ANP is produced and released from the atrial myocardium in response to wall stretching and results in vasodilatation, natriuresis, and inhibition of the renin–angiotensin–aldosterone system.6 Several studies have reported that the plasma concentration of ANP can be used to predict the severity of heart disease. In humans, increased ANP has been described in patients with left ventricular dysfunction, including acute myocardial infarction and dilated cardiomyopathy (DCM).6–9 In dogs, Asano et al10 reported that ANP is strongly correlated with pulmonary capillary wedge pressure (PCWP) and indicates the severity of mitral valve regurgitation. Thus, evaluating ANP concentrations is expected to facilitate estimation of the severity of heart disease or distinguish it from pulmonary disease in clinical practice.
The amino acid sequence of ANP in cats is similar to that of humans, dogs, cows, and horses (72, 76, 76, and 80% sequence homology, respectively).5 Therefore, evaluating plasma C-terminal ANP concentration in cats may be useful in estimating the severity and prognosis of heart disease. However, the basic and clinical implications of evaluating C-terminal ANP concentration in cats are still controversial. Thus, we investigated the relationship between plasma C-terminal ANP concentration and left atrial pressure (LAP) in healthy cats with volume overload, and compared the plasma C-terminal ANP concentration between normal cats and cats with cardiomyopathy.
Material and Methods
This study followed the Guidelines for Institutional Laboratory Animal Care and Use at the School of Veterinary Medicine at Kitasato University, Japan.
Two separate studies were performed.
Five adult mixed-breed cats of both sexes (1 male and 4 females) weighing 3.0–5.0 kg were used in this study. The cats were housed individually in cages and fed with commercial dry food with free access to water. All cats were determined to be normal by physical and echocardiographic examinations.
After sedation with butorphanol (0.2 mg/kg, IV) and atropine (0.025 mg/kg, SC), the cats were anesthetized with propofol (6.0 mg/kg, IV) and intubated. Anesthesia was maintained with 2.0% isoflurane in oxygen. The cats were positioned in left lateral recumbency. Respiratory rate was maintained with an artificial ventilator.a End-tidal PaCO2 was monitored and maintained between 35 and 45 mmHg, and heart rate was monitored with an electrocardiogram.b The chest was opened by a left thoractomy at the 4th or 5th intercostal space. The heart was exposed and suspended in a pericardial cradle. A 4 Fr fluid-filled catheter was placed into the left atrium and connected to a strain-gauge manometerb for LAP measurements. After completion of the procedures, a 20–30-min stabilization period was provided to establish baseline condition. Preload was increased by the IV infusion of acetated Ringer's solution at 100 mL/kg/h for 60 minutes via the cephalic vein, a modification of the dosage reported by Cheung et al.11 During IV infusion, we monitored the influence of volume overloading on hemodynamic variables and found mean LAP to be 10–15 mmHg above baseline. Hemodynamic measurements and blood sampling were performed at baseline at 10-min intervals. Blood samples were drawn from the jugular vein. After all examinations, cats were given furosemide (2 mg/kg, IV) and recovered from anesthesia.
Clinically healthy cats (n=8) were studied. These cats were adult mixed-breed cats, weighing 3.0–5.0 kg, including both sexes. Two of the 5 female cats were spayed. None of the 3 male cats was castrated. All cats were housed individually in cages as purpose-bred cats and fed with commercial dry food with free access to water. These cats were determined to be healthy on the basis of complete physical examinations, electrocardiography, and echocardiography. All cats were examined without anesthesia or sedation.
The study population consisted of 14 cats with cardiomyopathy. Owners provided informed consent before their cats participated in the study. The characteristics of the cats with cardiomyopathy are given in Table 1. The diagnosis of cardiomyopathy was based on complete physical examination, electrocardiography, plasma biochemical evaluation, thoracic radiography, and echocardiography. Cats with cardiomyopathy were diagnosed with hypertrophic cardiomyopathy (HCM; n=4), restrictive cardiomyopathy (RCM; n=4), DCM (n=2), or unclassified cardiomyopathy (UCM; n=4). Cats with HCM were diagnosed by echocardiography when the interventricular septum, left ventricular free wall in diastole, or both were >6 mm.12–14 In 2-dimensional (2-D) echocardiography, M-mode scanning was performed on the right parasternal short-axis view of the left ventricle, just above the papillary muscles. Cats with RCM were diagnosed by the presence of marked left atrial dilatation without concomitant myocardial hypertrophy, and a relatively normal left ventricular chamber.12,14,15 DCM was diagnosed when left ventricular end-systolic diameter was >14 mm and fractional shortening decreased (≤ 28%) by 2-D M-mode echocardiography of the right parasternal short-axis view.12,14,15 The diagnosis of UCM was made only after making certain that the myocardial abnormality did not fit any of the recognized disease classifications.12,15 Exclusion criteria for the study included known concurrent systemic illness other than cardiovascular disease, congenital cardiac disease, and previous long-term drug therapy for cardiac disease. Breeds included 10 mixed-breed cats, 1 American Shorthair, 1 Chinchilla, 1 Persian, and 1 Scottish Fold. Twelve cats were graded as having cardiac murmurs I–III and 2 cats had gallop rhythms. Cats were classified by New York Heart Association (NYHA) class as I–IV: 1 cat was NYHA class I, 2 were NYHA class II, 5 were NYHA class III, and 6 were NYHA class IV. Pleural effusion, pulmonary edema, or both were seen in 8 cats. Cats were divided into 2 groups on the basis of the presence of clinical signs of congestive heart failure (CHF) and the results of physical examination, electrocardiography, plasma biochemical evaluation, thoracic radiography, and echocardiography; 6 cats were negative for heart failure (CHF[−]) and 8 cats were positive for heart failure (CHF[+]). Blood samples were collected from the cephalic vein at initial diagnosis for measurement of plasma C-terminal ANP in both clinically healthy cats and cats with cardiomyopathy. After blood sampling and all procedures were completed, all cats were given medical treatment for heart disease.
Table 1. Comparison of the signalments between normal cats and cats with heart disease.
Normal (N = 8)
CHF(−) (N = 6)
CHF(+) (N = 8)
Data are presented median values (range).
P < .05 versus normal group.
P < .01 versus normal group.
CHF, congestive heart failure; BW, body weight; VHS, vertebral heart size; NYHA, New York Heart Association.
Blood was collected into tubes containing aprotinin and centrifuged at 1,500 ×g (4 °C) for 10 minutes. Plasma samples were stored at −70 °C until measurement. Plasma C-terminal ANP concentrations were determined by radioimmunoassay (RIA) for human α-ANPc according to the manufacturer's instructions.10 This assay is intended for the measurement of human C-terminal ANP from biological fluids; it contains mouse antihuman ANP antibody. It is known that the amino acid sequence of ANP in cats is highly homologous with that of humans.5 All assays were performed in duplicate. Precision studies done by the kit manufacturer indicated an interassay coefficient of variation of <10% at concentrations of 20, 108, and 501 pg/mL, respectively. The range of the standard curve was 0–2,000 pg/mL. The detection limit of the assay was 5–2,000 pg/mL. In human body fluids, cross-reactions with other ANP were not detected in the range from 5 to 2,000 pg/mL.
In study 1, plasma C-terminal ANP concentration was corrected for diluted rate of hematocrit values (Ht%) compared with baseline as follows: corrected C-terminal ANP concentration (pg/mL)=measured C-terminal ANP × (baseline Ht%/measured Ht%).
Transthoracic echocardiography was performed with an ultrasound unitd with a 12 MHz probe. The echocardiograms were analyzed by the commercial analysis software package supplied with the system.d The data were stored digitally and analyzed off-line by a single observer (Y.H.). The average of 3 cardiac cycles was calculated.
The left atrium to aorta (LA/Ao) ratio was acquired on a cardiac short-axis view. End-diastolic and end-systolic left ventricular internal diameters (LVIDd and LVIDs, respectively) were measured in the short-axis view and left ventricular fractional shortening (LVFS) was calculated.
Pulsed Doppler echocardiography was applied to measure transmitral inflow velocity in the 4-chamber view with the sample volume positioned at the tips of the mitral valve leaflets. Then, peak early (E wave) and late (A wave) diastolic transmitral inflow velocities and ratio of the E wave to A wave were measured. Aortic outflow Doppler measurements were made with the sample volume placed just below the aortic valve from an apical long axis view. Ejection time (ET) was measured from the onset to the end of the aortic outflow. The isovolumic relaxation time (IRT) was calculated by subtracting intervals between the peak of the R wave and the end of ET from intervals between the peak of the R wave and the onset of the E wave.11,16 The isovolumic contraction time (ICT) was calculated by subtracting ET and IRT from the intervals between cessation of the mitral valve A wave and the onset of the mitral valve E wave of the next cardiac cycle.11,16
In study 1, data were described as mean ± SD. The hemodynamic measurements and C-terminal ANP concentration were analyzed with repeated measures of analysis of variance. The significance of the differences between the mean values in the baseline and each condition was tested with the posthoc Tukey multiple comparison test. Correlation coefficients (r) and regression equations were calculated between corrected C-terminal ANP concentration and LAP. In study 2, data were described as median value (range). The data were analyzed with the Kruskal–Wallis test and Dunn's test was used for posthoc analysis. A value of P < .05 was considered statistically significant.
The hemodynamic measurements were markedly changed by volume overload, as described in Table 2. Heart rate and Ht% were decreased significantly from baseline (P<.01 and P<.001, respectively). Volume overload significantly increased the systolic LAP, mean LAP, diastolic LAP, and corrected C-terminal ANP concentration from baseline (P<.001, respectively). Corrected C-terminal ANP concentration was strongly correlated with the mean LAP (Y= 8.9X+ 27, r= 0.67, P<.001; Fig 1).
Table 2. Volume overload-related changes of hemodynamic measurements in study 1 (N = 5).
The echocardiographic measurements are described in Table 3. The E wave was significantly increased in the CHF(+) group compared with the normal cats (P<0.01). The LA/Ao ratio was significantly higher in the CHF(−) and CHF(+) groups compared with the normal cats (P<.05, P<.01, respectively). Compared with the normal cats, plasma C-terminal ANP concentration was significantly increased in the CHF(−) and CHF(+) groups (18.5 pg/mL [10–57] versus 111 pg/mL [78–300]; P<.05 and versus 228 pg/mL [95–585]; P<.01, Fig 2).
Table 3. Comparison of echocardiographic findings between normal cats and heart disease cats.
Data are presented Median values (range).
P < 0.05 versus normal group.
P < 0.01 versus normal group.
HR, heart rate; LVIDd, end-diastolic LV internal diameters; LVPWd, end-diastolic LV posterior wall thickness; LVSd, end-diastolic LV septum thickness; FS, fractional shortening; E wave, mitral early diastolic wave; A wave, mitral late diastolic wave; E/A ratio, ratio of E wave to A wave; LA/Ao, ratio of the left atrium to the aorta; ICT, isovolumic contraction time; IRT, isovolumic relaxation time; ET, ejection time; MPI, myocardial performance index.
In study 1, we demonstrated that volume overload led to an immediate increase in plasma C-terminal ANP in anesthetized healthy cats. ANP is one of the natriuretic peptides, which consists of 28 amino acids and is released from the atrium in response to wall stretching.5 It has been reported that ANP is released from the atrium in humans and dogs.17,18 Biondo et al reported that the distribution of ANP can be determined by immunohistochemistry in the atrium of cats.5,19 In addition, our data are consistent with previous findings that plasma ANP concentration was immediately increased by acute volume overloading in human patients with ischemic heart disease.20 In addition, plasma ANP concentration was significantly correlated with PCWP or left ventricular end-diastolic pressure.20 Furthermore, plasma C-terminal ANP concentration is significantly correlated with PCWP or LAP in dogs with heart disease.10 These reports support our data that the plasma C-terminal ANP concentration was significantly correlated with LAP in anesthetized healthy cats. This information may allow the prediction of cardiac filling abnormalities in cats.
In study 2, we investigated plasma C-terminal ANP concentration as a biomarker for the diagnosis and severity (ie CHF[−] or CHF[+]) of heart disease in cats with cardiomyopathy. Plasma C-terminal ANP concentration was significantly increased in cats with cardiomyopathy compared with normal cats. In humans, measurement of ANP concentration is useful for predicting the severity and prognosis of heart disease, and patients with high ANP concentrations have a poor prognosis.6,7,21 Dogs with occult DCM have significantly higher ANP concentrations than do normal dogs,2 which indicates the clinical utility of ANP measurement. In another report, high plasma ANP concentration (>95 pg/mL) was related to decreased survival time in dogs with heart disease, including DCM and mitral valve regurgitation, which indicates that plasma ANP concentration is a noninvasive predictor of survival in those dogs.22 Thus, our results indicate that the measurement of plasma C-terminal ANP may be useful in a clinical setting as a biomarker for the diagnosis of heart disease in cats. However, MacLean et al23 reported that although plasma N-terminal ANP (NT-ANP) in cats with HCM was higher than that in normal cats, the difference was not significant. This discrepancy may be explained by methodological differences as follows: the concentration of NT-ANP was determined by enzyme immunoassay and was evaluated in cats with subclinical heart disease in the previous study, whereas we determined the concentration of C-terminal ANP by RIA and evaluated C-terminal ANP in cats with moderate or severe heart disease.
Our study had several limitations. The RIA used in the present study was intended for determination of C-terminal ANP concentration in human biological fluids. It has been reported that the amino acid sequence in feline ANP is highly homologous to that of humans and dogs.5 The possibility that factors in feline plasma interfere with the assay or that feline peptides other than ANP cross-react with the assay was unknown. Our study was limited by small sample size: 5 normal cats in study 1 and 14 cats with cardiomyopathy in study 2. In study 1, we could not exclude the possibility that general anesthesia may have affected C-terminal ANP concentration in healthy cats. Complete autonomic blockade was not used in the present study, and reflex autonomic changes might have affected heart filling. In study 2, because it has been reported that plasma ANP in humans is affected by age,24 we have to interpret our results with caution. Cats with several forms of cardiomyopathy were enrolled in the present study, and therefore additional studies are required to define the relationship between C-terminal ANP and each individual heart disease. The relationship between C-terminal ANP concentration and severity or outcome in cats with heart disease is still unclear.
Our results showed that plasma C-terminal ANP concentration was strongly correlated with LAP in healthy cats. Furthermore, C-terminal ANP concentration was significantly higher in cats with cardiomyopathy compared with normal cats. These results suggest that measuring the C-terminal ANP concentration in cats has the potential to provide additional information in the diagnosis of heart disease.
Grant: This study is supported by the Kitasato University Research Grant for Young Researchers (No. 3128).
aKV-1a, Kimura Medical Instrument Co Ltd, Tokyo, Japan