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European Journal of Heart Failure 2006 8(2):179-186; doi:10.1016/j.ejheart.2005.07.010
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© 2006 European Society of Cardiology

Independent and incremental prognostic value of endogenous ouabain in idiopathic dilated cardiomyopathy

Maria Vittoria Pitzalisb, John M. Hamlync, Elisabetta Messaggioa, Massimo Iacoviellob, Cinzia Forleob, Roberta Romitob, Elisabetta de Tommasib, Paolo Rizzonb, Giuseppe Bianchia and Paolo Manuntaa,*

a Division of Nephrology, Dialysis and Hypertension, University "Vita-Salute" San Raffaele Hospital Via Olgettina 60, 20132 Milan, Italy
b Institute of Cardiology, University of Bari Bari, Italy
c Department of Physiology, School of Medicine, University of Maryland Baltimore, MD, USA

* Corresponding author. Tel.: +39 0226433891; fax: +39 0226432384. E-mail address: manunta.paolo{at}hsr.it


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Increased circulating levels of endogenous ouabain (EO) have been observed in some heart failure patients, but their long term clinical significance is unknown. This study investigated the prognostic value of EO for worsening heart failure among 140 optimally treated patients (age 50±14 years; 104 male; NYHA class 1.9±0.7) with idiopathic dilated cardiomyopathy. Plasma EO was determined by RIA and by liquid chromatography mass spectrometry, values were linearly correlated (r=0.89) in regression analysis. During follow-up (13±5 months), heart failure progression was defined as worsening clinical condition leading to one or more of the following: sustained increase in conventional therapies, hospitalization, cardiac transplant, or death. NYHA functional class, age, LVEF, peak VO2 and plasma levels of EO were predictive for heart failure progression. Heart failure worsened 1.5 fold (HR: 1.005; 95% CI: 1.001—1.007; p<0.01) for each 100 pmol/L increase in plasma EO. Moreover, those patients with higher plasma EO values had an odds ratio of 5.417 (95% CI: 2.044—14.355; p<0.001) for heart failure progression. Following multivariate analysis, LVEF, NYHA class and plasma EO remained significantly linked with clinical events. This study provides the first evidence that circulating EO is a novel, independent and incremental marker that predicts the progression of heart failure.

Key Words: Cardiac failure • Progression • Na pump • Ouabain-like factor

Received November 26, 2004; Revised May 3, 2005; Accepted July 14, 2005


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
There are many neurohormonal abnormalities associated with the progression of left ventricular dysfunction to heart failure [1], however, their precise roles remain unclear. This uncertainty [2,3] also applies to endogenous ouabain [EO], a mammalian steroid hormone [4], that is involved in sodium homeostasis [5,6] and blood pressure regulation [7]. Several studies have suggested that EO may have a primary role in causing cardiac dysfunction and failure. In a study in rats, chronic infusion of very low doses of ouabain to double the plasma concentration of EO, triggered a signal transduction pathway that produces cardiac hypertrophy [8]. In a second study, the young offspring of hypertensive patients had higher plasma levels of EO than the offspring of normotensive parents which were correlated with diastolic dysfunction [42]. In another study, in newly diagnosed patients with mild hypertension, plasma levels of EO were found to be bimodally distributed [9]. The low (normal) mode was similar to that of normotensive patients whereas the high mode had a median value almost twice the normal value. In the high EO mode patients, left ventricular mass and stroke volume were increased while heart rate was lower. In a fourth study of patients with more advanced hypertension, circulating levels of EO were directly related to both blood pressure and total peripheral resistance and inversely related to cardiac index [10].

These observations prompted us to investigate the prognostic value of plasma EO in patients with idiopathic dilated cardiomyopathy, on the assumption that EO may have a primary role in the progression of heart failure. These patients were chosen to avoid the confounding effects of systemic hypertension and cardiac ischemia. EO was measured using a well-established radio-immunoassay [11] in addition some samples underwent analysis by mass spectrometry for additional verification.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Patients
This was a prospective study of 140 consecutive patients with idiopathic dilated cardiomyopathy referred to our Institution as outpatients or for hospitalisation between January 1998 and March 2003.

In addition, 203 normotensive healthy subjects who attended the San Raffaele Hospital in Milan gave informed consent for blood sampling. Subjects with a medical history of myocardial infarction, heart failure, stroke, diabetes mellitus, liver disease, use of oral contraceptives, or the abuse of drugs or alcohol were excluded.

Idiopathic dilated cardiomyopathy was diagnosed on the basis of the patients' clinical history, a physical examination, 12-lead electrocardiography, chest radiography, echocardiography, left ventriculography and coronary angiography according to the WHO criteria [12]. All patients were in a clinically stable condition and were taking optimal therapy for at least three months at the time of study entry.

The study was approved by the local Ethics Committee and all patients gave written informed consent.

2.2. Echocardiographic examination
Mono and two-dimensional echocardiography recordings were obtained using a phased-array echo-Doppler system (Hewlett Packard Sonos 2500) equipped with a 2.5 MHz transducer. According to the recommendations of the American Society of Echocardiography [13], left ventricular end diastolic diameter (LVEDD) was obtained using a parasternal long axis view; left ventricle ejection fraction (LVEF) was calculated using Simpson's rule [11].

2.3. Cardiopulmonary exercise testing
117 patients underwent symptom-limited bicycle ergometer exercise testing with assessment of oxygen consumption (VO2) by mass spectrometry (Sensormedics System 2900, Anaheim, CA). The system was calibrated with a standard gas of known concentration before each test. The testing protocol consisted of 2 min of free pedalling followed by 20 W increments every 2 min at a constant pedal speed of 55-60 rpm. A 12-lead ECG was monitored continuously and recorded every minute for determination of heart rate and ST segment changes. Patients were encouraged to exercise to exhaustion, and all participants stopped exercise as a result of breathlessness and/or fatigue. The highest oxygen consumption (peak VO2) at peak exercise was measured during the last 30 s of symptom-limited exercise and expressed as millilitres per kilogram per minute.

2.4. Endogenous ouabain assay and liquid chromatography mass spectrometry
The blood samples were drawn after the subjects had rested in a supine position for at least 30 min, collected in tubes containing EDTA (1.5 mg/ml), and then centrifuged at 4 °C within 30 min. The plasma was transferred into plastic tubes and stored at –70 °C prior to analysis. The assay used an ouabain antiserum with low crossreactivity for digoxin (~4%), spironolactone (<0.01%), canrenone (<0.01%) and canrenoate (0.07%). In addition, all plasma samples were extracted by C-18 solid phase methods as previously described [14]. EO was selectively desorbed using low concentrations of acetonitrile so that digoxin, spironolactone, and its metabolites canrenone and canrenoate remained bound. Under these extraction conditions, the overall assay crossreactivity was minimal (digoxin<0.01%, spironolactone and related metabolites<0.0001%) so that EO could be determined with confidence in patients who were receiving digitalis and/or spironolactone. The dried sample extracts were reconstituted in water and used for EO radioimmunoassay as described previously [14]. Briefly, the detection limit was 25 pmol/L, and other standard curve parameters were: Kd 3.5±0.2 nmol/L, (inter assay CV 8%, intra assay CV 5%), Hill coefficient 1±0.01, lower control 150 pmol/L (inter assay CV 6%), high control 750 pmol/L (inter assay CV 3%). In addition, EO levels were assessed in four randomly selected patients by liquid chromatography mass spectrometry (LCMS). The LCMS analysis was performed using an Agilent 1100 capillary LC system linked to a Bruker Esquire ion trap mass spectrometer. Following injection of 1 ml equivalent of the extracted plasma sample, a gradient of acetonitrile in water was used to elute bound materials from the capillary LC column. The effluent was mixed with acetonitrile containing lithium carbonate and passed to the electrospray interface of the MS instrument. The column effluent was monitored for molecular ions whose mass to charge ratio matched that for lithiated EO (i.e., m/z 591). For MS-MS studies, molecular ions at m/z 591 were isolated and selected for collision induced dissociation (CID). The intensity of the product molecular ion corresponding to the lithiated aglycone of EO (i.e., m/z 445.4) was determined. Calibration was performed by injecting known amounts of ouabain over the linear range (25-500 fmol) of the LCMS combination and monitoring the retention time and intensity of the lithiated ouabagenin product ion at m/z 445.4. The EO content of the samples was obtained by interpolation. The mass spectrometry was performed in Baltimore by an operator who was blinded to both the clinical status of the patients and the RIA plasma value.

2.5. Follow-up
The patients were followed up in an outpatient setting, with scheduled visits every three months and clinical and instrumental examinations as required. The primary endpoint was the clinical progression of heart failure, which was prospectively defined as worsening of heart failure leading to a sustained increase in conventional medication (beta-blockers, diuretic, ACE inhibitor, AT1 inhibitor, digitalis), hospitalization, cardiac transplantation or death [16]. Data on deaths and hospitalizations were collected regardless of cause. Hospitalisations were classified for heart failure, and for cardiovascular or noncardiovascular reasons. Deaths were classified as cardiovascular or noncardiovascular; cardiovascular death was defined as death due to heart failure progression (caused by progressive hemodynamic deterioration) and as sudden death [17]. For patients who died outside hospital or in secondary centres, the relatives were interviewed about the terminal event and the related charts were collected from the referring physician or hospital.

2.6. Statistical analyses
Data are presented as means±SD. Following ANOVA, normal continuous variables were compared using the t-test; otherwise, the Mann-Whitney U test was used. Analysis of covariance was used to compare EO values between the groups with and without digitalis therapy. Frequencies were compared using Fisher's exact test. Relations between variables were assessed by using the Pearson correlation coefficient. The Cox proportional-hazards model was used to assess the association of the study variables with the events (hazard ratio and 95% confidence interval, CI, for risk factors are given). The hazard ratio for a continuous variable refers to the risk ratio per unit of the analysed variable. To assess the incremental prognostic value of the variables, an additional multivariate Cox Regression model was performed in which the studied variables were added sequentially in the same order in which they would be considered in clinical practice. Kaplan-Meyer cumulative survival curves were also constructed using the median value of plasma EO to dichotomise the study population into two groups. The tests were considered statistically significant when the p value was <0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
The clinical characteristics of the 140 patients enrolled in the study are shown in Table 1.


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  		Table 1 Clinical characteristics of patients with and without events during follow-up

 
3.1. Clinical correlates
Regression analyses showed that plasma EO was significantly correlated with NYHA functional class (r=0.38, p<0.001), systolic (r=–0.25, p<0.01) and diastolic blood pressure (r=–0.21, p<0.05), LVEF (r=–0.37, p<0.001), LVEDD (r=0.27, p=0.001) and peak VO2 (r=–0.33; p<0.001), but not with age or body mass index. Plasma EO levels were higher in patients who were taking digitalis therapy (396.43±187.45 vs. 211.59±71.35 pmol/L; p<0.001).

3.2. Prognostic significance of EO
During follow-up (30±14 months) the following events were observed: five patients had worsening of heart failure symptoms, which led to an increase in conventional therapy; 20 patients were hospitalised for heart failure or pulmonary oedema; three patients underwent urgent cardiac transplantation, one patient died following hospitalisation for heart failure. Three patients died suddenly. The clinical characteristics of patients with and without events are shown in Table 1.

As shown by the univariate analysis in Table 2, NYHA functional class, age, LVEF, LVEDD, peak VO2 and plasma levels of EO were highly predictive for heart failure progression. EO was predictive for heart failure progression in patients with (HR: 1.003; 95% CI: 1.001-1.005; p<0.05) as well as in patients without (HR: 1.006; 95% CI: 1.002-1.011; p<0.01) digitalis therapy, implying worsening heart failure at a rate of 1.5 times per 100 pmol/L increase in plasma EO. Table 3 shows the plasma EO levels in 203 healthy subjects according to age (young/old) and in patients with idiopathic dilated cardiomyopathy grouped according to NYHA class. Those patients with the worst heart failure (NYHA class 3) were found to have higher circulating EO (p<0.001). Moreover, when we considered patients with plasma EO values above or below the median level (233 pmol/L), those with higher values had an HR of 5.417 (95% CI: 2.044-14.355; p<0.001) for heart failure progression. The Kaplan-Meier curves for patients with plasma EO values above or below the median level are shown in Fig. 1 and illustrate the more rapid decline of patients with high EO levels.


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  		Table 2 Univariate analysis-predictor (Cox Proportional Hazard Ratio)

 


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  		Table 3 Circulating EO levels in healthy subjects according to age and patients with idiopathic dilated cardiomyopathy grouped by NYHA Class

 


Figure 1
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  		Fig. 1 Kaplan-Meier cumulative events reflecting worsening heart failure in two groups of patients with dilated cardiomyopathy where circulating EO was either above or below the median value of the study population.

 
Circulating EO, when considered as a continuous variable, remained significantly associated with heart failure progression after adjustment for age, LVEDD, LVEF, NYHA functional class and digitalis therapy (Table 4). The prognostic significance of EO was also evident after peak VO2 was taken into consideration in those patients for whom this parameter was measured (Table 4). As shown in Fig. 2, the interactive stepwise procedure revealed the power of the various relationships to predict major events in hierarchic order (age; age and NYHA class; age, NYHA class and LVEF; age, NYHA class, LVEF and EO).


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  		Table 4 Multivariate stepwise Cox proportional hazard analysis for progression of heart failure among patients with dilated cardiomyopathy

 


Figure 2
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  		Fig. 2 Results of multivariate stepwise analysis. The power (R2) of the various relationships to predict major events in hierarchic order is shown.

 
3.3. Endogenous ouabain immunoassay and liquid chromatography mass spectrometry
LCMS was used to confirm the presence of EO in the sample extracts from four patients whose EO was also determined by radioimmunoassay. Fig. 3 presents the data obtained for one of the patients and shows the extracted MS-MS ion current chromatogram for product molecular ions with m/z 445.4 following CID. The lithiated molecular ion of the EO aglycone was observed as a large ion current at 52.6 min. The retention time of the endogenous molecular ion under the solvent gradient conditions used was similar to that for the lithiated ouabagenin product ion in this system (not shown). Fig. 4 shows the product ion scan resulting from CID of the parent ion of EO at 52.6 min. As expected, only residual traces of the parent molecular ion (m/z 591) were observed (large arrow) whereas product molecular ions at m/z 445.4 and 427.3 corresponding to the lithiated aglycone of EO and its singly dehydrated counterpart, respectively, were present. The EO content of the samples determined by LCMS and the ouabain RIA were correlated (r=0.89) in linear regression analysis. None of the above mentioned molecular ions were observed when extracts of water were used instead of plasma samples.


Figure 3
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  		Fig. 3 LCMS/MS capillary ion chromatogram for a plasma extract from a patient with dilated cardiomyopathy. Shown are the elution characteristics of positively charged molecular product ions whose mass to charge ratio is equivalent to the lithiated aglycone of ouabagenin (m/z 445.4). The specific current at 52.6 min is the lithiated aglycone of endogenous ouabain.

 


Figure 4
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  		Fig. 4 MS/MS scan of molecular product ions resulting from collision induced dissociation of the lithiated molecular ion of human endogenous ouabain. The scan was performed on the column eluate at 52.6 min. The inset shows the correlation (r=0.89) between plasma EO determined by RIA and LC-MS/MS from four patients.

 
Neither digoxin nor spironolactone was present in any significant way in the sample extracts used for the EO immunoassay. Native plasma samples doped with 10 nM digoxin (concentrations 5-10 times the normal digitalizing dose) had EO immunoreactivity after extraction (mean±SEM, 265±18 pM) that was indistinguishable from their (water doped) controls (273±28 pM, n=10). Moreover, no ouabain immunoreactivity was detected in extracted water (replacing plasma) irrespective of whether it was doped with 10 nM digoxin or not (i.e., values less than assay threshold~5 pmol/L). Similar results were obtained when spironolactone was used up to 500 nmol/L. Thus, it can be concluded that these drugs do not significantly affect the EO immunoassay under the experimental conditions of the present study.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The primary finding of this study is that increased plasma levels of EO have an independent and incremental prognostic value in identifying patients with idiopathic dilated cardiomyopathy, who are likely to have worsening heart failure during follow-up. The prognostic value of EO described in this work is new, and was independent of demographic, clinical and echocardiographic data and digitalis administration. Our observations provide more evidence of a significant role for EO in the complex scenario of heart failure and may have an impact on therapy.

Several observations, including the natural history of the relationship between EO and cardiac changes in humans, together with data from experimental animals, suggest that EO contributes to the rapid progression of cardiac failure [2,3,7,8]. In agreement with data from Gottlieb [2], EO levels were higher in patients with poor hemodynamic status and these patients were found to have the worst prognosis. High circulating EO augments the activity of the sympathetic nerves [2,3,18-20], the renin angiotensin system [21,22], cytokine production [23,24], increases peripheral vascular resistance [9]. These maladaptive effects of EO are likely to contribute to the poor prognosis in patients with high circulating levels of this steroid [25].

EO may impact on various cardiac functional indices used to define the initial status of patients, as well as the progression of cardiac failure. For this reason, the independent effect of EO may be greater following adjustment for other indices of cardiac function. Indeed, when EO was added to the model in Table 4 in which other parameters were already included, the global R2 value increased significantly (global R2=44.8). Thus, EO provides additive prognostic information to the commonly used criteria.

In our patients, left ventricular dysfunction was not due to ischemia or systemic hypertension and the predictive role of EO was independent from these and other recognised risk factors linked with the progression of heart failure. Moreover, as an incremental risk factor, EO seems an attractive prognostic tool for the identification of patients at the highest risk, especially when used in combination with the parameters commonly available in clinical practice. The informative nature of EO is noteworthy considering that our evaluations were performed while patients were in a stable clinical condition and with optimal medical treatment.

Another interesting point is the observation that EO levels were significantly higher (+88%) in patients who were receiving digitalis therapy. This is noteworthy for two reasons. First, the observation does not reflect digoxin interference in the assay for EO. The combination of differential extraction as well as the limited cross-reactivity of the EO immunoassay for digoxin effectively excludes the participation of digitalis glycosides [10,13]. Due to the design of this study, it was not possible to interrogate the LC ion chromatograms for evidence of digoxin because this drug has a mass to charge ratio above the scan range used to probe for EO (400-650 m/z). Nevertheless, the absence of digoxin in extracted plasma samples suggests that we would not have expected to see molecular ions of digoxin to any significant degree. In addition, in the four samples that were available for MS analysis, a search for protonated and lithiated molecular ions of spironolactone was negative throughout the entire LC run. Therefore, even when digoxin and/or spironolactone are present in native plasma, neither drug appears to be present in the extracted samples used for the EO immunoassay. Secondly, the prognostic value of EO was independent of digitalis. This is of great interest because it suggests that cardiac glycosides such as digoxin and EO may have different functional consequences in heart failure and because the Digitalis Investigation Group (DIG) found no overall impact of digoxin on mortality. However, the DIG study did not consider EO in its design [26] and whereas heart failure patients with low and normal circulating levels of EO may benefit from digoxin, those with higher EO levels should, in all likelihood, not be given digitalis [25,26]. Nevertheless, the present study provides the first evidence that the combination of digoxin and high EO concentrations in the human circulation is associated with remarkably poor prognosis in the heart failure setting.

The mechanism of higher circulating EO levels in digitalised patients likely involves altered secretion and/or clearance of EO. The kidneys are the primary clearance route for ouabain and digoxin [27,28] with digoxin being actively transported into the tubular lumen via p-glycoprotein [29]. The basolateral membranes of human and rat proximal tubular cells actively accumulate digoxin and ouabain [30] and as the former inhibits ouabain uptake, digitalis therapy is likely to reduce the renal clearance of EO. In addition, digoxin may augment EO secretion by reducing the feedback inhibition of EO on biosynthesis [31]. The relative importance of these mechanisms in digitalized patients requires further investigation.

Several biomarkers have been described for the progression of heart failure [32,33]. Among these, brain natriuretic peptide (BNP) [34,35] appears to be especially valuable in identifying patients that are likely to have a more rapid decline in cardiac function. In patients with heart failure, it seems likely, although not investigated here, that BNP and EO may be elevated in the same patients. Moreover, cardiac glycosides augment the secretion of atrial peptides, including BNP, although this effect appears to be relatively modest [36]. A recent review of 19 studies indicated that the risk of death in all cause heart failure increased 35% for each 100pg/ml increase in BNP [32]. We did not measure atrial peptides in this study so direct comparison of the prognostic significance of EO versus BNP is not feasible. Moreover, the patients in our study were only followed for 2 years during which time the increased number of events in the high EO group was clinically significant but did not include a high number of fatalities. Therefore, more prolonged studies of the prognostic value of EO are needed to address the question of mortality.

Nevertheless, the prognostic value of EO in patients with dilated cardiomyopathy likely reflects a direct functional role for EO. For example, novel pharmacological agents, along with digoxin antibody fragments that bind ouabain and EO, block ouabain-induced increases in renal sympathetic nerve activity, peripheral vascular resistance and blood pressure in rats [37,38]. Moreover, immunological neutralization of EO in rats with heart failure prevented the impairment of baroreflex function, sympathetic hyperactivity, and dilation and dysfunction of the left ventricle post-myocardial infarction [39,40]. These studies imply that EO has functional significance in the progression of heart failure [32].

In this study, we used LCMS to prove that EO was present in the available sample extracts. The LCMS paradigm differs from prior work with protonated ions [4,41] in that the more stable lithiated adducts of ouabain and EO were monitored. Under dual MS (MS/MS) conditions, dissociation of the lithiated parent ion led to a diagnostic product ion representing the lithiated aglycone of EO (i.e., 445.1 m/z). The appearance of this product ion at the appropriate LC retention time shows for the first time that EO can be specifically detected in the human circulation and quantitated (Fig. 3) from small clinically relevant volumes of plasma. Moreover, these analytical results support the overall integrity of several studies that have used our immunoassay methods.

In conclusion, among patients with otherwise stable idiopathic dilated cardiomyopathy, high circulating levels of EO identify those individuals predisposed to progress more rapidly to heart failure. The prognostic value of EO appears to be independent of other commonly used parameters. The mechanism of action that underlies the prognostic significance of EO as well as its interactions with other biomarkers and digoxin requires further study.


   Acknowledgements
 
This work was in part supported by grants from Ministero Università e Ricerca Scientifica of Italy (FIRB Grant RBNE01724C_001 to GB and PRIN Grant 2004069314_01 to GB) and from Ministero della Salute (ICS 110.4/RF02353), and the USPHS (HL075584) to JMH.


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

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