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European Journal of Heart Failure 2001 3(2):165-171; doi:10.1016/S1388-9842(00)00132-X
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© 2001 European Society of Cardiology

Increased circulating levels of ouabain-like factor in patients with asymptomatic left ventricular dysfunction

Silvana Balzana,*, Danilo Negliaa, Sergio Ghionea, Giuseppina D'Ursob, Maria Carmela Baldacchinoa, Umberto Montalib and Antonio L'Abbatea

a CNR Institute of Clinical Physiology via Savi no. 8, 56126 Pisa, Italy
b Department of Science of Man and Environment, University of Pisa Pisa, Italy

* Corresponding author. Tel.: +39-50-583-277; fax: +39-50-553-461. E-mail address: balzan{at}ifc.pi.cnr.it (S. Balzan).


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Background: Much evidence has been accumulated that human plasma contains digitalis-like factor(s) with Na/K ATPase inhibitor properties. Increased concentrations of ouabain-like factor (OLF) have been reported in patients with moderate to severe hypertension and in patients with overt congestive heart failure due to dilated cardiomyopathy.

Aim: The presence of circulating OLF has not been investigated in borderline to mild hypertension or in the early stage of dilated cardiomyopathy.

Methods and Results: The study population consisted of 18 normal volunteers, 24 patients with borderline to mild hypertension, 47 patients with asymptomatic left ventricular dysfunction (ALVD) due to dilated cardiomyopathy and 26 patients with cardiac arrhythmias but normal left ventricular function. OLF values (pM ouabain equivalent) were assayed in extracted plasma, using a radioimmunoassay for ouabain. OLF was, respectively, 29.4 ± 20.6 pM in normal controls, 39.1 ± 23.8 pM in hypertensives, 35 ± 18 pM in patients with cardiac arrhythmias, 52.3 ± 25.8 pM in ALVD patients not treated with digoxin and 64.6 ± 29.6 pM in ALVD patients treated with digoxin. Patients with ALVD, both treated and not treated with digoxin, had OLF significantly higher (P < 0.05) than all the other groups. In patients with ALVD no correlation between OLF and left ventricular ejection fraction was observed. In the hypertensive group no correlation between OLF and both diastolic and systolic pressure was found.

Conclusion: Increased concentrations of OLF were observed in patients with left ventricular dysfunction due to dilated cardiomyopathy, before the occurrence of overt heart failure, suggesting that OLF may be an early marker of the disease.

Key Words: Ouabain • Dilated cardiomyopathy • Hypertension • Cardiac arrhythmias

Received May 9, 2000; Revised August 2, 2000; Accepted October 12, 2000


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Several lines of evidence suggest the presence in mammalian tissue and plasma of one or more endogenous inhibitors of Na+/K+ ATPase with digitalis-like activity. In particular, in 1991 Hamlyn et al. [1] described a factor in human plasma that was indistinguishable from ouabain [ouabain-like factor (OLF)]. Endogenous OLF inhibits the sodium–potassium pump. In vitro it has cardiac and vascular actions, including positive inotropic and vasoconstrictive effects, similar to plant derived ouabain [2]. The availability of sensitive and specific immunoassays for OLF [3–5] allowed the investigation of its potential role in disease states. Above normal concentrations of OLF have been reported in approximately 50% of subjects with moderate to severe hypertension [6–8] as well as in patients with overt congestive heart failure mainly due to dilated cardiomyopathy [9]. The possible pathophysiologic role of the elevated OLF levels in these patients is still debated. In hypertensive patients a cause–effect relationship for elevated OLF levels and both higher blood pressure and increased left ventricular mass has been suggested [8]. In heart failure the observation that patients with the highest OLF concentrations had the lowest cardiac indexes argued against the hypothesis that lack of endogenous ouabain may contribute to the cardiac dysfunction; it rather suggested that increased OLF could be a compensatory response to the abnormal physiology of heart failure [9].

In all the available clinical studies, OLF determination has been mainly performed in patients with advanced diseases including severe hypertension and dilated cardiomyopathy with overt heart failure. Circulating OLF was not determined in patients with milder forms of these diseases where increased concentrations could be associated with the disease processes independently from secondary homeostatic adjustments. Moreover, different patient populations have been examined with different techniques for OLF determination preventing a direct comparison of different populations.

The purpose of this study was to assess whether circulating OLF levels are increased in patients with mild hypertension (HT) or asymptomatic left ventricular dysfunction (ALVD) due to dilated cardiomyopathy. Accordingly the measurements were done using the same radioimmunoassay technique in: (1) normotensive volunteers; (2) patients with mainly mild essential hypertension and normal ventricular mass; (3) normotensive patients with cardiac arrhythmias or conduction disturbances but normal ventricular function; and (4) patients with ALVD due to dilated cardiomyopathy. Groups number 1 and number 3 were studied as controls for the HT and ALVD populations.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Patient population
OLF was measured in plasma samples of 115 subjects. Eighteen subjects were healthy normal volunteers recruited from our Institute. Twenty-four were patients who were referred to our hypertension outpatient clinic because of reported mild to moderate elevation of blood pressure. According to their mean ‘casual’ clinic blood pressure obtained from three measurements by an automatic recorder (Dinamap, Critikon) in a seated position and following the classification of JNC VI [10], six subjects had high-normal blood pressure (systolic blood pressure 130–139 mmHg or diastolic blood pressure 85–89 mmHg), 14 had stage 1 (mild) hypertension (140–159 mmHg or 90–99 mmHg) and four subjects had stage 2 (moderate) hypertension (160–179 mmHg or 100–109 mmHg). All were untreated and none had echocardiographic evidence of increased left ventricular mass (left ventricular mass index <150 g/m2 in men and <120 g/m2 in women) [11]. The clinical characteristics of the normotensive and hypertensive subjects are reported in Table 1. Twenty-six patients had accidentally discovered cardiac arrhythmias, normal blood pressure and normal ventricular function (left ventricular ejection fraction >50%) by equilibrium radionuclide angiography. Cardiac arrhythmias included complex ventricular arrhythmias (ventricular ectopic beats, Lown class≥III) in 18 patients and bundle branch block (BBB) in eight patients (left BBB in seven, right BBB in one). Forty-seven patients had ALVD attributed to dilated cardiomyopathy. These patients were without symptoms of overt heart failure (NYHA class I–II) and had come to medical attention because of accidentally discovered cardiac arrhythmias, consisting of complex ventricular arrhythmias in 17 patients and of bundle branch block in 18 patients (left BBB in 17, right BBB in one), or because of accidentally discovered LV dysfunction at echocardiography (12 patients). They had normal blood pressure, depressed systolic function at equilibrium radionuclide angiography (LVEF<50%) without evidence of restrictive or hypertrophic myocardial disease. The clinical characteristics and the functional data obtained by equilibrium radionuclide angiography in arrhythmic subjects and in patients with ALVD are reported in Table 2. At the time of the study, 13 patients with ALVD were under digoxin treatment. In all patients with cardiac arrhythmias or ALVD, other cardiac diseases, including hypertension, valvulopathy, ischemic heart disease and congenital heart disease were excluded by routine cardiological evaluation. In 37 out of 47 ALVD patients an invasive angiographic study was also performed to exclude coronary artery disease showing angiographically normal coronary arteries in all. Systemic diseases including diabetes, endocrine diseases, rheumatic diseases and neoplasty were excluded in all patients by routine clinical evaluation.


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  		Table 1 Clinical characteristics of normotensive and hypertensive subjectsa

 


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  		Table 2 Clinical characteristics of arrhythmic patients (ARR) and patients with asymptomatic left ventricular dysfunction (ALVD)a

 
2.2. Purification of plasma samples
Peripheral blood samples were drawn in heparinized tubes, centrifuged for 15 min at 3000 rev./min at 4°C, separated and stored at –20°C until the assay. Plasma (1 ml) was diluted 1:2 with water containing 0.1% TFA, centrifuged at 3000 rev./min for 0.5 h and passed through SepPak C18 cartridges previously activated with 5 ml of acetonitrile (0.1% TFA) and 10 ml of water (0.1% TFA). After the cartridges were washed with 10 ml of water, elution was performed with 3 ml of acetonitrile (25%) and dried under vacuum at room temperature. In separate experiments, both [3H]ouabain and [3H]digoxin (0.5 pmol) was added to normal adult plasma (1 ml) and then passed through SepPak C18 cartridges as described above. The activity in the eluate was expressed as a percent of the total radioactivity present in samples not passed through SepPak C18 cartridges.

2.3. Radioimmunoassay (RIA)
Anti-ouabain antiserum was raised in two rabbits as previously described [12]. Only compounds having a structure very similar to ouabain had a significant cross-reactivity with the antisera (% IC50 ouabain: digoxin 3.8%, digitoxin 36%, bufalin 2.7%). Rabbit anti-ouabain antiserum was diluted 1.5x104 in 10 mM phosphate buffer containing BSA (5 g/l), NaCl (154 mM), 0.1% Tween 20 and sodium azide (15 mM). RIA was done on samples after SepPak extraction and on ouabain standards as previously described [5]: the samples were dissolved in 200 µl of the same buffer as above and incubated with antiserum (50 µl) and with [3H]ouabain (50 µl; NEN 1 µC/ml) for 24 h at 4°C. In order to separate free from bound ouabain, an affinity resin was employed in which antidigoxin Fab fragments (Digibind Wellcome) were bound to Sepharose [13]. This resin was also able to bind, in addition to digoxin, ouabain and OLF [13,14]. The packed resin was diluted four times in Tris buffer 20 mM (pH 7.4) and 200 µl were added and incubated under stirring for 30 min at 4°C. The mixture was then centrifuged and the supernatant counted by liquid scintillation. This method gave sample values in ouabain-equivalents which correlated with those obtained from the inhibitory activity assay of 86Rb uptake in erythrocytes [5].

2.4. Statistics
The interpolation of the dose–response curves, the precision profile and all sample values were calculated by a computer program [15]. Group data were expressed as ouabain equivalent (o.e.)/l of plasma (mean±S.D.). Comparison was made by one-way ANOVA and post-hoc multiple comparisons (Scheffe's F-test) using the Statview 2 statistics package on an Apple Macintosh computer. Regression analysis was performed by the least squares method.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
Calibration ouabain RIA curve and (inset) precision profile obtained from 15 consecutive assays is shown in Fig. 1. Assay sensitivity was 15±7 fmol/tube with a mean IC50 of 392±56 fmol/tube. [3H]Ouabain was recovered in the fraction with 25% acetonitrile (97±2%), whereas [3H]digoxin was undetectable in this fraction and was completely eluted by 45% acetonitrile. OLF levels were similar in males (45.2±26.5 pM, n=81) and females (37.4±22.9 pM, n=34) and no correlation was observed with age (r=0.054, ns).


Figure 1
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  		Fig. 1 Ouabain RIA calibration curve and interassay precision profile (inset) from 15 consecutive assays. Data represent the mean±S.D.

 
Fig. 2 shows the scatterplot of OLF levels in the four groups of subjects considered: OLF mean plasma concentrations were, respectively, 29.0±18.3 pM (o.e.) in normal controls, 39.1±23.8 pM in patients with mild hypertension, 34.5±18.8 pM in patients with cardiac arrhythmias, 52.3±25.3 pM in patients with ALVD not treated with digoxin and 64.6±29.6 pM in patients with ALVD treated with digoxin. ANOVA showed a significant difference between groups (P=0.0001) and post-hoc comparison demonstrated that the patients with ALVD, both treated and not with digoxin had OLF levels significantly higher (P<0.05) compared with all the other groups. In the 13 patients with ALVD who were receiving digoxin, the mean OLF was not significantly different (P=0.16, ns) from ALVD patients without digoxin treatment.


Figure 2
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  		Fig. 2 Plasma ouabain-like factor (OLF) concentrations in individual subjects: normals (N), borderline hypertensive subjects (HT), patients with various cardiac arrhythmias but normal ventricular function (ARR) and patients with asymptomatic left ventricular dysfunction not treated with digoxin (ALVD) and treated with digoxin (ALVD*). Mean OLF concentrations ±S.D. are also shown.

 
There was no correlation between OLF values and both diastolic or systolic blood pressure values either in the hypertensive group (r=0.032 and r=0.32, ns, respectively) or in the hypertensive and the three normotensive groups combined (r=0.05 and r=0.05, ns, respectively). No correlation was observed with left ventricular mass.

In the groups of patients with cardiac arrhythmias or ALVD no difference in OLF values was found between patients with bundle branch block or with complex ventricular arrhythmias (Fig. 3). In these two groups combined, OLF levels were inversely correlated with LVEF (r=0.33, P<0.01) but this relation was not observed in the group of ALVD patients alone (r=0.06, ns) (Fig. 4). No correlation was present between OLF levels and either LV volume (r=0.02, ns) or cardiac output (r=0.05).


Figure 3
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  		Fig. 3 Bar graphs show ouabain-like factor (OLF) and ejection fraction (EF) in patients with cardiac arrhythmias (ARR) and asymptomatic left ventricular dysfunction (ALVD), respectively, with bundle branch block (BBB) or complex ventricular arrhythmias (CVA). NO, ALVD patients without cardiac arrhythmias.

 


Figure 4
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  		Fig. 4 Relation between ouabain-like factor (OLF) concentrations and left ventricular ejection fraction (LVEF) in patients with asymptomatic left ventricular dysfunction (ALVD).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The present study was designed to test the hypothesis that increased concentrations of endogenous ouabain could be detected not only in patients with moderate-severe hypertension or advanced dilated cardiomyopathy but also in milder forms of these diseases, i.e. before the occurrence of observable organ damage or subjective or clinical signs of heart failure.

The main result of the present study is the demonstration of increased plasma concentrations of endogenous ouabain in patients with ALVD due to dilated cardiomyopathy. In this population circulating levels of OLF were not related with the extent of left ventricular dysfunction and were independent from the presence of complex ventricular arrhythmias or conduction disturbances. In fact in patients presenting with these arrhythmias but showing normal ventricular function OLF levels were similar to those in normal controls. The present results suggest that in dilated cardiomyopathy increased concentrations of endogenous ouabain are associated with the myocardial disease process because they can be demonstrated in early stage disease when the secondary effects of heart failure can be excluded.

A second conclusion from the present study is that circulating OLF levels are not increased in patients with mild hypertension without cardiac hypertrophy. These data confirm previous reports which showed increased endogenous ouabain levels only in patients with more severe hypertension and organ damage.

4.1. Determination of endogenous ouabain
Among the variety of endogenous substances with digitalis-like properties which have been reported to be present in mammalian plasma, the one at present best characterized is a factor very similar or even identical to ouabain; named therefore ouabain-like factor (OLF). Several methods have been developed to detect this factor by employing anti-ouabain antibodies. However, the results are difficult to be compared because the values of OLF vary widely, ranging from low levels (5–160 pM) [4–6,16–18] to higher values (300–1000 pM) [7,19]. These discrepancies could be due to differences in the recovery by the extraction procedures and/or in the specificity of the antibodies employed. A further possibility which has been recently proposed is that OLF may be intrinsically unstable and give rise, to a variable and unpredictable extent, to inorganic complexation products with different biological and immunological properties [20].

These methodological difficulties hinder cross laboratory comparison of results obtained in different patient populations with different techniques. In the present study we used the same method to detect circulating OLF levels in different patient populations. The assay was sensitive (15±7 fmol/tube with a mean IC50 of 392±56 fmol/tube) and specific since it allowed a 97±2% recovery of [3H]ouabain in the fraction with 25% acetonitrile whereas [3H]digoxin was undetectable in this fraction. OLF levels were similar in males and females and no correlation was observed with age.

4.2. Ouabain and hypertension
A possible pathophysiologic role of OLF in arterial hypertension has been hypothesized since the beginning of the studies on endogenous digitalis-like factors in the early eighties and various studies have reported increased levels in various animal models [21,22] and in patients with this disease [6,7,19,23]. Recently, Manunta et al. [8] in a study on a large number of untreated normotensive and hypertensive subjects provided evidence that, compared with normotensive controls, hypertensive subjects have a wide scatter of OLF levels, suggesting a bimodal distribution, and that the subjects with an OLF above the normal range also have higher blood pressure levels and an increased left ventricular mass.

In the present study, OLF values in hypertensive patients were not significantly different than in normal controls and were not correlated with blood pressure levels. The present population was completely different from previously reported study groups since 20 out of 24 patients had mild hypertension and in no subject left ventricular hypertrophy could be documented. In this mildly hypertensive group pressure values could be highly variable ranging during the day from normal to borderline abnormal levels. This phenotypic heterogeneity might have obscured the relationship between OLF and pressure levels. However, in the scatter of OLF values in the hypertensive group no bimodal pattern could be recognized and no correlation with blood pressure values could be found also when hypertensive and normotensive groups were considered together. Thus, the present results stand against a pathophysiologic role of endogenous ouabain in the early development of mild hypertension. It may be consistent with the hypothesis that increased OLF levels are a risk factor for more severe forms of hypertension associated with organ damage.

4.3. Ouabain and dilated cardiomyopathy
Despite the well known role for over a century of digitalis drugs in the treatment of heart failure surprisingly few studies have investigated the circulating levels of endogenous ouabain in this syndrome [9,21]. This is even more surprising if one considers that the two available clinical studies reported higher than normal values of OLF in heart failure and that in one of these studies a significant association was found between increasing levels of OLF and decreasing levels of heart function. Most of the patients included had idiopathic dilated cardiomyopathy as the etiologic cause of heart failure rising the interesting question whether increased levels of endogenous ouabain participated to the complex homeostatic response to the abnormal physiology of heart failure or were directly associated with the disease process causing myocardial dysfunction.

The present study was centered on the hypothesis that abnormal regulation of endogenous ouabain could precede the development of heart failure being directly associated with myocardial dysfunction due to cardiomyopathy. Accordingly, we carefully selected a population of patients, without signs or symptoms of heart failure, with left ventricular dysfunction attributed to an early cardiomyopathic process in the absence of other causes. Data obtained in these patients were compared with those obtained in normal controls, in hypertensives and in subjects with normal ventricular function similarly presenting with cardiac arrhythmias. Patients with ALVD had higher levels of OLF than the other groups. The fact that increased concentration of OLF are present in patients with ALVD but not in patients with similar presentation but normal ventricular function indicate OLF as a potential marker of a disease which involves the myocardium. It should be also considered that OLF values were inversely related with LVEF in the two populations combined and that this correlation was not present in the population of ALVD patients alone. This observation may suggest that the increased OLF levels mark the disease process which causes left ventricular dysfunction but are not directly related with the severity of myocardial impairment. In fact, no correlation was found between OLF levels and other hemodynamic or functional variables such as the cardiac output and the degree of ventricular dilation. These results apparently contrast with previous data obtained in overt heart failure, in patients after acute myocardial infarction [24] or with non-obstructive hypertrophic cardiomyopathy [25]. In these populations, higher pressure and/or volume overload could have caused a higher dependence of OLF circulating levels on hemodynamic variables. As a matter of fact the pathophysiologic role of increased OLF plasma concentrations in dilated cardiomyopathy is yet unknown. It is interesting to note that recently Schwinger et al. [26] in addition to a reduced expression of {alpha}1 and {alpha}3 isoform of Na+/K+ ATPase in human cardiomyopathic heart, observed also a reduced activity of Na+/K+ ATPase. It could be speculated that either increased OLF levels result from an homeostatic response to reduced Na+/K+ pump sites concentration/activity [27] or in turn they are responsible for a down-regulation of these sites or the decrease of their activity with obvious effects on heart function. A further hypothesis stands on the known effects of ouabain to cause coronary and systemic vascular vasoconstriction [28]. We recently demonstrated that patients with early stage dilated cardiomyopathy without overt heart failure frequently show a severe impairment of myocardial perfusion, due to decreased coronary microvascular vasodilating capability which is associated with progression of the disease and worse prognosis [29,30]. The possible association of increased OLF values and decreased coronary vasodilating capability in early stage dilated cardiomyopathy will deserve more attention in the future.

4.4. Ouabain and cardiac arrhythmias
OLF levels were measured in patients with cardiac arrhythmias and normal ventricular function. No significant difference was found compared to normals. Moreover, no significant difference in OLF values was found between patients with complex ventricular arrhythmias or conduction disturbances both in ALVD and arrhythmic populations. Digitalis compounds are known to give rise to arrhythmias and a possible arrhythmogenic role of a digitalis-like factor has been suggested by Bagrov et al. [31] who observed that saline-pre-treated rats with myocardial ischemia had high levels of digoxin-like immunoreactivity and an increased incidence of ventricular arrhythmias, which declined after administration of antidigoxin antibodies. Since antidigoxin antibodies neutralize also ouabain and OLF [14] these results could indicate a role of OLF in ventricular arrhythmias. The present results do not confirm this hypothesis in this particular clinical setting. Further experimental and clinical studies are needed to clarify this issue.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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M. Ferrandi, I. Molinari, P. Barassi, E. Minotti, G. Bianchi, and P. Ferrari
Organ Hypertrophic Signaling within Caveolae Membrane Subdomains Triggered by Ouabain and Antagonized by PST 2238
J. Biol. Chem., August 6, 2004; 279(32): 33306 - 33314.
[Abstract] [Full Text] [PDF]

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S. A. Jortani, S. D. Prabhu, and R. Valdes Jr
Strategies for Developing Biomarkers of Heart Failure
Clin. Chem., February 1, 2004; 50(2): 265 - 278.
[Abstract] [Full Text] [PDF]

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