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European Journal of Heart Failure 2008 10(12):1186-1191; doi:10.1016/j.ejheart.2008.09.013
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© 2008 European Society of Cardiology

Sympathetic activation in congestive heart failure: Reproducibility of neuroadrenergic markers

Guido Grassia,b,c,*, Gianbattista Bollab, Fosca Quarti-Trevanoa, Francesca Arenarea, Gianmaria Brambillaa and Giuseppe Manciaa,b,c

a Clinica Medica, Dipartimento di Medicina Clinica, Prevenzione e Biotecnologie Sanitarie, Università Milano-Bicocca Ospedale San Gerardo, Monza (Milan), Italy
b Centro di Fisiologica Clinica e Ipertensione Milan, Italy
c Istituto Auxologico Italiano Milan, Italy

* Corresponding author. Clinica Medica, Ospedale S Gerardo, Via Pergolesi 33, 20052 Monza (Milano), Italy. Tel.: +39 039 2333357; fax: +39 039 322274. E-mail address: guido.grassi{at}unimib.it (G. Grassi).


   Abstract
Top
 Abstract
1. Methods
 2. Results
 3. Discussion
 References
 
Objective: To assess the reproducibility of the two markers of adrenergic drive, venous plasma norepinephrine (NE) and efferent postganglionic muscle sympathetic nerve activity (MSNA), in reflecting the sympathetic activation characterizing congestive heart failure (CHF).

Methods and measurements: In 19 CHF male normotensive patients (mean age: 53.0±2.1 years, NYHA classes II and III, left ventricular ejection fraction 35.9±2.9%), blood pressure (BP, Finapres), heart rate (EKG), plasma NE (HPLC assay) and MSNA (microneurography, peroneal nerve) were measured in two experimental sessions separated by a week interval. At each session, three NE samples were obtained and NE reproducibility between sessions was assessed by considering single NE samples or averaging 2–3 samples.

Results: While MSNA values showed a highly significant correlation between sessions (r = 0.85, P<0.001), NE values based on a single blood sample evaluation did not correlate with each other (r = 0.41, P = NS). NE correlation coefficients improved and achieved statistical significance when average data from 2 and 3 blood samples were examined (r = 0.54 and r = 0.57, P<0.02 for both).

Conclusions: In CHF, MSNA displays a better reproducibility pattern than plasma NE. The reproducibility of the NE approach, however, can be improved by performing the assay on multiple blood samples.

Key Words: Heart failure • Sympathetic activity • Plasma norepinephrine • Muscle sympathetic nerve traffic • Muscle sympathetic nerve activity

Received April 28, 2008; Revised July 30, 2008; Accepted September 24, 2008

Congestive heart failure (CHF) is characterized by a marked sympathetic activation which is 1) directly proportional to the severity of the CHF state [1-6], 2) of similar magnitude in CHF of ischaemic and idiopathic dilated aetiology [7], 3) affects several vascular circulations such as the cerebral, the coronary, the muscle and the renal ones [6,7-11], 4) potentiated when other clinical conditions also characterized by adrenergic overdrive, such as hypertension, obesity and metabolic syndrome, are concomitantly present [12,13], and 5) relevant for patient's prognosis because it is inversely related to survival rate [2,14-16]. The adrenergic overdrive characterizing the CHF syndrome is detectable by both indirect and direct approaches to assess sympathetic tone, such as plasma norepinephrine (NE) assay, NE spillover technique as well as microneurographic recording of efferent postganglionic muscle sympathetic nerve activity (MSNA) [1-11].

In several studies, however, and particularly in those carried out in mild to severe CHF, NE has frequently failed to detect the increase in adrenergic cardiovascular drive documented by approaches directly assessing human sympathetic function [7,12,13]. This is also the case for some of the studies aimed at investigating the effects of non-pharmacological or pharmacological interventions on adrenergic function [17,18]. Among the reasons suggested to explain the reduced ability of NE to reliably reflect sympathetic drive in CHF, a likely one is the limited reproducibility of the approach [19], as suggested by data obtained in essential hypertension, another condition characterized by a hyperadrenergic state [20]. Whether a limited reproducibility of NE is also present in CHF has been never investigated. In addition, whether and to what extent in CHF this limitation can be overcome by performing the NE assay on multiple blood samples, as we have recently shown to be the case in essential hypertension [21], has never been examined.

The present study was designed to address the two above mentioned issues. To achieve this aim, multiple NA samples were obtained at two different experimental sessions, reproducibility was compared with that of MSNA, which has been shown to faithfully reflect sympathetic activity in both the short- and the long-term period in other conditions [20].


   1. Methods
Top
 Abstract
 1. Methods
2. Results
 3. Discussion
 References
 
1.1. Study population
Patients were recruited into the study if they had 1) an echocardiographic left ventricular ejection fraction between 30% and 45%, 2) a body mass index <26 kg/m2, 2) normal (<140/90 mm Hg) blood pressure (BP) values, 3) no history of smoking, excessive alcohol consumption or major non-cardiovascular disease, including diabetes mellitus, 5) no alteration in renal function or ultrasonographic evidence of carotid artery thickening or plaques, 6) no evidence of metabolic syndrome and sleep apnoea syndrome, 7) no myocardial infarction or stroke in the 12 months preceding the study, 8) no history of regular exercise habit or involvement in physical training programmes and 9) a cardiac sinus rhythm. All patients were receiving treatment with furosemide, angiotensin converting enzyme inhibitors or angiotensin II receptor antagonists and beta-blockers in various combinations and dosages. With the exception of beta-blockers, which were withdrawn 7 days before the first study session until the end of the second experimental session, all other drugs were maintained throughout the study period without altering their daily dosage. In each patient, BP was measured 3 times in the sitting position with a mercury sphygmomanometer, taking the first and fifth Korotkoff sound to identify systolic and diastolic values respectively. Left ventricular end-diastolic and end-systolic diameters were obtained from an echocardiogram performed in M-mode after selection of the measurement section by a B-mode scan. The echocardiographic data, which were used to assess left ventricular ejection fraction [22], were calculated by a single operator. The within operator coefficient of variation of left ventricular diameter data was 5.8%. The study protocol was approved by the Ethics Committee of the institutions involved. All of the patients agreed to participate after being informed of the nature and purpose of the study.

1.2. Measurements
Multiunit recording of MSNA was obtained from a microelectrode inserted in a peroneal nerve posterior to the fibular head, as reported previously [6-8,12,13,17,18]. Integrated nerve activity was monitored by a loud-speaker, displayed on a storage oscilloscope (model 511A, Tektronix), and continuously recorded with BP and heart rate on an ink polygraph. The muscle nature of MSNA was established according to criteria described in previous studies [6-8,12,13,17,18], and nerve recording was accepted only if the signal:noise ratio was >3. In the two experimental sessions, MSNA was quantified as bursts incidence corrected for heart rate values (bursts per 100 heart beats). This quantification has been shown to provide reproducible values that differ only by 3.8% when assessed twice in the same session by a single investigator [6]. Plasma NE was measured by high performance liquid chromatography [23,24] from a blood sample taken from a venous cannula positioned in an antecubital vein. The method enables the detection of NE changes of 5 pg/ml and the mean coefficient of variation of values obtained from the same sample is 5.4%. These data for sensitivity are similar to those reported by others [25]. During each experimental session, BP was monitored by a finger photopletismographic device (Finapres 2300, Ohmeda) capable of providing accurate beat-to-beat systolic and diastolic values [6-8,12-15,17,18]. Heart rate was monitored beat-to-beat by a cardiotachometer triggered by the R wave of an EKG lead. Respiration rate was monitored by a strain-gauge pneumograph positioned at midchest level.

1.3. Protocol and data analysis
MSNA measurements were carried out in the morning after an overnight fast. The first venous blood sample for plasma NE determination was withdrawn with the patient resting in a supine position and 30-min after insertion of the cannula. BP, heart rate and MSNA were then continuously measured over a 60-minute baseline period. Second and third blood samples for plasma NE assay were taken at the 30th and 60th minutes of the recording period, respectively. The same procedure was repeated during the second experimental session which was performed after a time interval of one week, during which patients were asked not to modify their home therapeutic regimen, lifestyle or physical activity levels.

Data were analyzed by a single investigator who was blinded to the study design and session from which the data originated. For each patient, the 60-minute recording period was divided for data analysis into 3 subperiods, each lasting 20 min. Values of BP, MSNA and heart rate were averaged separately 1) for each subperiod and 2) for two and three consecutive subperiods. The same procedure was followed for the 3 sets of NE data. Individual values of BP, MSNA, heart rate and NE measured in the baseline period were averaged for the whole group, expressed as means±SEM and compared by two-way analysis of variance. Reproducibility of the MSNA and NE data obtained in the two experimental sessions was tested by means of the Pearson's correlation coefficient and the Bland-Altman Plot [26]. A P<0.05 was taken as the minimal level of statistical significance.


   2. Results
Top
 Abstract
 1. Methods
 2. Results
3. Discussion
 References
 
The study included 19 male patients with heart failure of either ischaemic (n=13) or idiopathic (n=6) aetiology. All patients were in New York Heart Association (NHYA) class II (n=13) or III (n=6). The main anthropometric and haemodynamic characteristics of the study population are shown in Table 1.


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  		Table 1 Demographic, anthropometric and haemodynamic variables in the study population (n=19)

 
Fig. 1 shows that the average values of systolic BP, diastolic BP, heart rate and MSNA were almost superimposable in the two experimental sessions of the present study. This was also the case for left ventricular ejection fraction and end-diastolic left ventricular diameter (data not shown). Average NE values were somewhat lower in the second experimental session, which was performed 1 week after the first session. As shown in Fig. 2, the correlation between MSNA values was extremely high between the data acquired in the two different experimental sessions (upper panels), a further slight increase being detected when average data obtained by the different subperiods were examined (lower panels). In contrast, the corresponding correlation between plasma NE values never achieved statistical significance (Fig. 3, upper panels). However, the correlation was statistically significant when average data derived from two and more so from three samples were examined (Fig. 3, lower panels). The reproducibility of the MSNA and NE data was confirmed by the Bland-Altman plots. In the total population sample, the mean difference in MSNA values between the different recording subperiods was small (1.5 bs/100 heart beats), in contrast to what seen for the mean difference of NE values (53 pg/ml). The magnitude of the NE difference, however, was reduced by 53% when average NE data were considered. In both the two experimental sessions, MSNA and NE displayed a weak and non significant correlation with each other (r=0.33, P=NS), and this was the case even when average data were considered (at best r=0.38, P=NS).


Figure 01
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  		Fig. 1 Systolic (S) blood pressure (BP), diastolic (D) blood pressure, heart rate (HR), muscle sympathetic nerve activity (MSNA) and plasma norepinephrine (NE) values measured in different subperiods of the two experimental sessions. Data are shown as mean±SEM from 19 heart failure patients. B/min: beats/minute; bs/100 hb: bursts/100 heart beats. Asterisk (*) refers to the statistical significance (P<0.05) between different NE values.

 


Figure 02
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  		Fig. 2 Correlations between individual MSNA values recorded in the two experimental sessions, taking into account the first sample (1st), the second sample (2nd), the third sample (3rd) or the average of two (1st-2nd or 2nd-3rd) or all three samples (1st-2nd-3rd). For symbols and explanations see Fig. 1.

 


Figure 03
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  		Fig. 3 Correlations between individual NE values recorded in the two experimental sessions, taking into account the first sample (1st), the second sample (2nd), the third sample (3rd) or the average of two (1st-2nd or 2nd-3rd) or all three samples (1st-2nd-3rd). For symbols and explanations see preceding figures.

 

   3. Discussion
Top
 Abstract
 1. Methods
 2. Results
 3. Discussion
References
 
The present study provides three pieces of new information about the sympathetic overdrive characterizing mild to moderate CHF. First, it shows that MSNA values recorded in NYHA class II CHF patients during two different experimental sessions show a highly significant correlation. Second, it documents that this is not the case for NE values which fail to display any significant relationship when evaluated in the two different time windows of the present study. Finally, it shows that the limited reproducibility of NE in reflecting the elevated adrenergic drive which characterizes the CHF state can be improved by performing the determination of the plasma neurotransmitter levels on multiple samples rather than on a single sample. Taken together these findings allow us to conclude that, in CHF 1) microneurographic assessment of sympathetic cardiovascular drive has a better reproducibility profile than the one characterizing NE assay and 2) the multiple sampling approach used in the present study facilitates improved reproducibility of plasma NE as an adrenergic marker.

The present study was not designed to investigate why MSNA has a better reproducibility profile than NE in CHF. The following hypotheses can be suggested, however. It can be speculated, for example, that while MSNA recording depends on a single major determinant, i.e. central sympathetic nerve firing rate, several other processes are involved in determining circulating plasma NE levels [27,28]. These include the release as well as the reuptake of the adrenergic neurotransmitter from nerve endings and the clearance process to which NE is exposed at the peripheral neural level [28,29]. All of these steps (particularly the last one) are affected by the haemodynamic alterations characterizing CHF [28], such as the reduction in cardiac output and thus the decrease in tissue perfusion pressure making random variations in plasma circulating NE levels more prone to take place. It can also be speculated, however, that the scanty reproducibility pattern of plasma NE may depend on the fact that the neurohumoral alterations of CHF involve several substances and systems which interfere with adrenergic function. This is the case for activation of the renin-angiotensin-aldosterone system, because elevated circulating angiotensin II levels may interfere with peripheral sympathetic neurotransmission and the NE reuptake process [30]. This is also the case for the insulin resistance state frequently detected in CHF [31], because the concomitant hyperinsulinaemic state triggers vasodilation and thus alterations in the tissue clearance of NE [32].

Three final considerations on the results of the present study deserve to be made. Firstly, although improved by the multiple sampling approach, the correlation coefficients between NE values observed in the two different experimental sessions were still much lower than the MSNA ones. This suggests that the NE approach, even when based on multiple sampling, cannot be regarded as being as capable of fully reflecting adrenergic drive as MSNA. This may be particularly the case when assessing the acute sympathetic responses to therapeutic interventions or haemodynamic adjustments. Secondly, only a weak and non significant correlation was found between heart rate and NE (r=0.19, P=NS), even when the multiple sampling approach was used. This finding, coupled with the evidence that this was also the case for MSNA (r=0.32, P=NS), supports the conclusion of a previous study by our group that in heart failure, hypertension and obesity heart rate are unable to reflect the adrenergic activation seen with microneurographic MSNA recording and/or plasma NE assay [33]. It is likely that this depends on the fact that heart rate is modulated not only by sympathetic but also parasympathetic influences, which are altered in CHF [34]. Finally, in the present study we examined NYHA class II and III CHF patients treated with different drugs. With the exception of beta-blocking agents, which were withdrawn one week before the study for a 2 week period until the performance of the second study, all other drugs were maintained for the whole period between the two studies, with no changes in dosage. This makes it unlikely that pharmacological treatment could have affected the results of the present study and the relationships we found between different sympathetic markers.

In conclusion, the present study shows that in CHF, NE reproducibility can be improved by performing the assay on multiple blood samples, thus making circulating levels of this neuroadrenergic transmitter more capable of reflecting the adrenergic overdrive typical of the CHF state.


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

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