MB Andrew J Hogarth

Alan F Mackintosh

David A S G Mary

PhD. M B Chb

Gender-Related Differences in the Sympathetic Vasoconstrictor Drive of Normal Subjects

Keywords: autonomic nervous system, gender, regional blood flow, vasoconstriction

The risk of cardiovascular disease has been linked to sympathetic activation and its incidence is known to be lower in women than in men. However, the effect of gender on the sympathetic vasoconstrictor drive has not yet been established. We planned to find out whether there is a gender difference in muscle sympathetic nerve activity (MSNA) and blood flow, and to determine the mechanisms involved.

We examined 68 normal subjects, 34 women and 34 men, matched for age, BMI and waist circumference. MSNA was measured as the mean frequency of single units (s-MSNA) and as multi-unit bursts (m-MSNA) from the peroneal nerve simultaneously with its supplied calf muscular blood flow (CBF).

Women had lower (P=0.0007) s-MSNA (24±2.0 impulses/100 cardiac beats) than men (34±2.3 impulses/100 cardiac beats), and a greater baroreceptor reflex sensitivity controlling efferent sympathetic nerve activity than men. The sympathetic activity was inversely and directly correlated respectively to CBF (P=0.03) and calf vascular resistance (CVR) (P=0.01) in men only. The responses of an increase in CVR to cold pressor and isometric handgrip tests were significantly smaller in women (P=0.002) than in men despite similar increases in efferent sympathetic nerve activity.

Women had a lower central sympathetic neural output to the periphery, the mechanism of which involved differences in central and reflex control, as well as a lower vasoconstrictor response to this neural output. It is suggested that this may partly explain the observed lower incidence of cardiovascular events in women compared to men.

Introduction

The level of arterial blood pressure and the incidence of cardiovascular disease, including hypertension, in middle-aged populations have been reported to be lower in women than in men [START_REF] Lerner | Patterns of coronary heart disease morbidity and mortality in the sexes: a 26-year follow-up of the Framingham population[END_REF][START_REF] Wiinberg | 24-h ambulatory blood pressure in 352 normal Danish subjects, related to age and gender[END_REF][START_REF] Burt | Prevalence of hypertension in the US adult population. Results from the Third National Health and Nutrition Examination Survey, 1988-1991[END_REF][START_REF] Mosca | Cardiovascular disease in women. A statement for healthcare professionals from the American Heart Association[END_REF][START_REF] Smulyan | Comparative effects of aging in men and women on the properties of the arterial tree[END_REF][START_REF] Reckelhoff | Gender differences in the regulation of blood pressure[END_REF]. However, despite the widely accepted relationship of sympathetic activation to the pathogenesis and cardiovascular complications of hypertension [START_REF] Mancia | Sympathetic activation in the pathogenesis of hypertension and progression of organ damage[END_REF][START_REF] Esler | The sympathetic system and hypertension[END_REF], the influence of gender on the sympathetic vasoconstrictor drive has not yet been established.

For instance firstly, there have been no published reports designed primarily to investigate gender-related differences in the levels of resting muscle sympathetic nerve activity (MSNA). Also, the information that is available from studies in which results have been obtained in men and women have been separately presented showing women to have either lower [START_REF] Ng | Age and gender influence muscle sympathetic nerve activity at rest in healthy humans[END_REF][START_REF] Jones | Gender differences in muscle sympathetic nerve activity: effect of body fat distribution[END_REF][START_REF] Jones | Gender does not influence sympathetic neural reactivity to stress in healthy humans[END_REF][START_REF] Matsukawa | Gender difference in age-related changes in muscle sympathetic nerve activity in healthy subjects[END_REF][START_REF] Shoemaker | Gender affects sympathetic and hemodynamic response to postural stress[END_REF][START_REF] Narkiewicz | Gender-selective interaction between aging, blood pressure, and sympathetic nerve activity[END_REF] or similar [START_REF] Matsukawa | Gender difference in age-related changes in muscle sympathetic nerve activity in healthy subjects[END_REF][START_REF] Narkiewicz | Gender-selective interaction between aging, blood pressure, and sympathetic nerve activity[END_REF][START_REF] Ray | Effect of gender on vestibular sympathoexcitation[END_REF][START_REF] Tank | Baroreflex regulation of heart rate and sympathetic vasomotor tone in women and men[END_REF][START_REF] Fu | Effects of gender and hypovolemia on sympathetic neural responses to orthostatic stress[END_REF] levels of resting MSNA relative to young and older men respectively.

Secondly, there has been no information regarding gender-related differences in the operation of peripheral sympathetic vasoconstrictor drive as represented by the relationship between MSNA and simultaneously measured calf muscle blood flow (CBF) and resistance (CVR). This is important because there are reports showing that such a relationship is influenced by local vaso-active, hormonal, and structural factors [START_REF] Folkow | Physiological aspects of primary hypertension[END_REF][START_REF] Freedman | Sex differences in peripheral vascular adrenergic receptors[END_REF][START_REF] Skarphedinsson | Sympathetic nerve traffic correlates with the release of nitric oxide in humans: implications for blood pressure control[END_REF]. Indeed, it is well known that levels of MSNA do not necessarily correlate to those of arterial pressure between individuals [START_REF] Skarphedinsson | Sympathetic nerve traffic correlates with the release of nitric oxide in humans: implications for blood pressure control[END_REF][START_REF] Sundlöf | Human muscle nerve sympathetic activity at rest. Relationship to blood pressure and age[END_REF][START_REF] Greenwood | Single unit sympathetic discharge: quantitative assessment in human hypertensive disease[END_REF].

We therefore planned to find out whether the mean frequency of efferent muscle sympathetic activity and calf muscular blood flow in normal women are different from those in matched normal men, and to determine the mechanisms involved. For this purpose, we simultaneously used microneurography directly to quantify efferent MSNA from the peroneal nerve and standard venous occlusion plethysmography to measure CBF during the steady state, cold pressor and isometric handgrip tests.

Methods

Subjects

We prospectively examined 68 normal Caucasian subjects. They comprised 34 women and 34 men. All had similar sedentary occupational status and dietary habits including a sodium intake of ≈400mmol/d, and none was engaged in exercise training.

They were screened by history and physical examination, and none was obese or had evidence of hypertension, arrhythmia, neuropathy or chronic conditions that may influence the autonomic nervous system. Twelve of the women were post-menopausal, while 6 men and 7 women gave a history of smoking. Arterial blood pressure was defined on the basis of the average of at least 3 sphygmomanometer readings, and all subjects had normal levels of systolic and diastolic arterial pressure [START_REF] Williams | British Hypertension Society. Guidelines for management of hypertension: report of the fourth working party of the British Hypertension Society, 2004-BHS IV[END_REF]. The subjects of the two groups were matched during recruitment according to age, body mass index, and waist circumference. The investigation conformed to the principles outlined in the Declaration of Helsinki and was approved by the St. James's University Hospital Ethics Committee, with all subjects providing informed written consent.

General Protocol

Microneurographic and haemodynamic measurements were obtained in an identical manner for all subjects during each session, as has previously been reported in detail [START_REF] Greenwood | Single unit sympathetic discharge: quantitative assessment in human hypertensive disease[END_REF][START_REF] Scott | Sympathetic nerve hyperactivity is associated with increased peripheral vascular resistance in hypopituitary patients with growth hormone deficiency[END_REF]. All investigations were performed under similar conditions between the hours of 09:00 and 12:00, and subjects were asked to have had a light breakfast and to empty their bladder before commencing the study. They were instructed to avoid nicotine and caffeine products for 12 hours, as well as alcohol and strenuous exercise for 24 hours prior to the investigation. During each session, the subjects were studied in the semisupine position when data attained a steady state for at least 30 minutes. Measurements were made in a darkened laboratory in which the temperature was constant between 22 and 24°C. Resting arterial blood pressure was measured from the arm, using a mercury sphygmomanometer. Changes in heart rate and arterial pressure were monitored and recorded, using a standard electrocardiogram and a Finometer device (FMS, Arnhem, The Netherlands, TPD Biomedical Instruments).

Microneurography

Post-ganglionic muscle sympathetic nerve activity (MSNA) was recorded from the right peroneal nerve, simultaneously with the other data as previously described [START_REF] Greenwood | Single unit sympathetic discharge: quantitative assessment in human hypertensive disease[END_REF][START_REF] Scott | Sympathetic nerve hyperactivity is associated with increased peripheral vascular resistance in hypopituitary patients with growth hormone deficiency[END_REF].

The neural signal was amplified (x50,000); then, it was either filtered (bandwidth of 700-2000 Hz) and integrated (time constant 0.1 sec) for the purpose of generating bursts representing multi-unit discharge, or left intact to examine raw action potentials. The output of action potentials and bursts from this assembly was passed to a PC-based dataacquisition system (LabView, National Instruments Corp., Austin, TX, United States), which digitised the acquired data at 12,000 samples/second (16 bits).

Muscle sympathetic nerve activity (MSNA) was differentiated from skin sympathetic activity and afferent activity, as previously described [START_REF] Greenwood | Single unit sympathetic discharge: quantitative assessment in human hypertensive disease[END_REF][START_REF] Cui | Baroreflex modulation of muscle sympathetic nerve activity during cold pressor test in humans[END_REF]. Single units (s-MSNA) in the raw action potential neurogram were obtained by adjusting the electrode position, whilst using fast monitor sweep, and on-line storage oscilloscope to confirm the presence of consistent action potential morphology [START_REF] Greenwood | Single unit sympathetic discharge: quantitative assessment in human hypertensive disease[END_REF][START_REF] Cui | Baroreflex modulation of muscle sympathetic nerve activity during cold pressor test in humans[END_REF]. Only vasoconstrictor units were accepted and examined, the criteria of acceptance being appropriate responses to spontaneous changes in arterial pressure during verification by a preliminary Valsalva manoeuvre and isometric handgrip exercise. During the Valsalva manoeuvre, sympathetic nerve activity increased during the latter part of phase-II and/or phase-III and decreased during phase-IV (respectively corresponding to the decrease and increase of arterial pressure). During the isometric handgrip exercise, performed using a dynamometer (MIE Medical Research Ltd., Leeds, UK), a delayed increase of sympathetic nerve activity was observed. In addition, simultaneous measurement of calf vascular resistance confirmed the vasoconstrictor function of the observed neural activity.

Analysis was performed independently off-line, using dedicated software based on the LabView system (National Instruments Corp., Austin, TX, USA). This allowed electronic superimposition of units from the raw action potentials to establish their same morphology (s-MSNA). Then, an electronic discriminator window was used objectively to count those s-MSNA spikes with consistent morphology and a threshold discriminator was used to count the R-waves of the electrocardiogram. The mean frequency of s-MSNA was quantified over one minute and over 100 cardiac beats, to avoid any interference by the length of the cardiac cycle [START_REF] Sundlöf | The variability of muscle nerve sympathetic activity in resting recumbent man[END_REF]. The multi-units bursts of MSNA (m-MSNA) were identified by inspection when the signal-to-noise ratio was greater than 3, and were counted and quantified in a similar manner to s-MSNA. The variability of repeated measurements of two minute segments of recordings of s-MSNA units and MSNA bursts spanning a period of 30 minutes or those of two impalements performed within 60 minutes did not exceed 10%, in terms of twice the 95% confidence intervals around individual differences relative to the mean of the repeated measurements [START_REF] Greenwood | Single unit sympathetic discharge: quantitative assessment in human hypertensive disease[END_REF].

Calf blood flow

Calf blood flow (CBF) was obtained simultaneously with microneurography, using an automated mercury-in-silastic (Whitney) strain gauge venous occlusion plethysmograph (D.E. Hokanson Inc., Bellevue, WA, USA). The strain gauge was placed around the widest circumference of the left calf region, and chosen to be 2-3 cm smaller than the calf circumference, such that it was applied under slight tension to the calf. Venous occlusion was effected by inflating a contoured thigh cuff (Model CC-22, D.E. Hokanson Inc., Bellevue, WA, USA), placed around the left thigh, to about 60 mmHg or 20 mmHg below the pre-determined diastolic arterial blood pressure, whichever was the lesser. The DC output from the plethysmograph was passed to a chart recorder (APC Medical Ltd., Welwyn Garden City, Herts., UK) utilising heat sensitive paper, so that a graphic record of change in limb volume could be produced.

During measurement of CBF, the left foot region was excluded by inflating a paediatric cuff placed around the ankle, to levels greater than the pre-determined systolic arterial blood pressure.

CBF was obtained typically at 3 recordings per minute during periods of steady state conditions. The average of the recordings was expressed in units of ml.100ml. - 1 min. -1 . The intra-observer reproducibility of CBF measurement in this laboratory, obtained as twice 95% confidence interval of differences between repeated withinsession plethysmography, amounted to 2.4% of the value of the measurement. Arterial blood pressure was simultaneously and continuously measured, and its average value was divided by the average CBF, to obtain calf vascular resistance (CVR), which was expressed in arbitrary units.

Other measurements

Responses of haemodynamic variables and sympathetic nerve activity to formal isometric handgrip exercise (IHG) and cold pressor (CPT) tests were measured. The former was performed at 30% of a pre-determined maximal voluntary contraction for two minutes and the latter by dipping the subject's hand into cold water with a temperature of less than 4 °C for at least one minute or until discomfort was felt.

Baseline and recovery data were taken for 1 minute prior to, and after each of the two tests. CPT was performed by all women and 32 men, while IHG was performed by 33 women and 31 men. The responses of sympathetic nerve activity and its vascular effect were obtained in 33 women and 31 men during CPT, and 30 women and 32 men during IHG; in the remaining subjects, it was not possible to obtain the activity because of interference by changes in leg muscle tension and inability to tolerate cold exposure.

Responses to isometric handgrip exercise were derived from the differences between data obtained during the second minute of the exercise, when sympathetic activity is known to increase [START_REF] Mark | Microneurographic studies of the mechanisms of sympathetic nerve responses to static exercise in humans[END_REF], and the average of those obtained during baseline and recovery periods. Responses to cold pressor test were derived as the differences between data obtained during the last 30 seconds of exposure to cold, when the occurrence of discomfort is known to be accompanied by an increase of sympathetic activity [START_REF] Kregel | Sympathetic nervous system activity during skin cooling in humans: relationship to stimulus intensity and pain sensation[END_REF], and the average of those obtained during baseline and recovery periods.

Baroreceptor reflex sensitivity controlling the heart period mainly through vagal effects (BRS-hp/sbp) and that controlling s-MSNA (BRS-sna/dbp) were obtained by a standardised Valsalva manoeuvre and measured independently off-line, using the LabView software. Subjects performed the manoeuvre to between 40 and 50 mmHg for 15 seconds. BRS-hp/sbp was obtained in all subjects, while BRS-sna/dbp was obtained in 28 women and 28 men. To obtain the BRS-hp/sbp, the time interval during which the rise of arterial blood pressure occurred in stage IV of the Valsalva manoeuvre was identified and each systolic arterial blood pressure value and its corresponding heart period (phase 0) and the succeeding one (phase 1) were analysed. BRS-hp/sbp was calculated from the slope [START_REF] Airaksinen | Valsalva manoeuvre in the assessment of baroreflex sensitivity in patients with coronary artery disease[END_REF][START_REF] Kautzner | Arterial baroreflex sensitivity assessed from phase IV of the Valsalva maneuver[END_REF] of the best significant linear relationship between the systolic arterial blood pressure and its heart period (phase 0) or the succeeding one (phase 1). BRS-sna/dbp was calculated in a similar manner by relating the diastolic arterial pressure values to the number of s-MSNA corrected for the length of the heart period over nine beats following each index arterial pressure. A close relationship of sympathetic nerve activity to short-term changes in diastolic arterial blood pressure has previously been observed [START_REF] Sundlöf | Human muscle nerve sympathetic activity at rest. Relationship to blood pressure and age[END_REF][START_REF] Kamiya | Static handgrip exercise modifies arterial baroreflex control of vascular sympathetic outflow in humans[END_REF][START_REF] Cui | Baroreflex modulation of muscle sympathetic nerve activity during cold pressor test in humans[END_REF].

Statistical analysis

Unpaired Student t tests were used to assess differences of data and absolute responses between the two groups. Percentage responses relative to control values were compared using the Mann-Whitney test. The least square technique was used for assessing the linear relationship between variables. Values of P < 0.05 were considered statistically significant. Data were presented as mean±SEM.

Results

The two groups were matched according to the design of the investigation in respect of age, body mass index (BMI), waist circumference and heart rate (Table 1).

Men were taller and had slightly higher arterial blood pressure values than women.

Compared to the group of men, women had significantly lower indices of sympathetic nerve activity (Fig. 1), lower BRS-hp/sbp and higher BRS-sna/dbp (Fig. 2).

No significant group differences were found between women and men regarding CVR (41±2.0 and 44±2.4 units; P>0.30) or CBF (2.3±0.08 and 2.4±0.13 ml.100ml. -1 min. -1 ; P>0.36). However, in men the indices of sympathetic nerve activity were positively correlated to CVR (at least r>0.38, P<0.002) and negatively correlated to CBF (at least r>-0.32, P<0.035), with no such significant correlations in women (respectively at least r<0.29, P>0.058 and r<-0.194, P>0.136). Figure 3 depicts the relationship between these variables in men and women.

The responses to CPT of the two groups are depicted in Fig. 4. Both groups of women and men had similar responses of heart rate, mean arterial blood pressure and sympathetic nerve activity to CPT. The respective percentage changes for women and men for heart rate (10±1.5% and 8±1.4%), mean arterial pressure (22±2.2% and 20±1.7%), m-MSNA (86±17.2% and 53±8.9%) and s-MSNA (120±20.4% and 103±21.9%) were not significantly different (at least P>0.20). However, the response of increase of CVR was significantly smaller in women than in men (Fig. 4); similarly its percentage increase was less (P<0.004) in women (46±5.9%) than in men (88±17.1%).

In respect of the responses to IHG of both groups, women had significantly smaller increases in heart rate and arterial blood pressure (Fig. 5). The percentage increases of heart rate (9±1.3%) and mean arterial pressure (14±1.4%) in women were smaller respectively (P<0.001 and P<0.001) than those in men (21±2.7% and 27±2.6%). Both women and men groups had similar responses of sympathetic activity to IHG (Fig. 5), though in terms of percentage changes the women showed greater increase (P<0.03) of m-MSNA (113±21.4%) than men (57±8.6%). The percentage increase of s-MSNA was also greater in women (195±35.4%) than in men (57±8.6%), though this was not statistically significant (P>0.07). However, the increase in CVR was smaller in women than in men (Fig. 5) and its percentage increase in women (28±3.0%) was smaller (P<0.001) than in men (69±9.6%).

Discussion

Our study has demonstrated in normal women and men that there were genderrelated differences in the level of MSNA, its vascular effect and its baroreceptor reflex control. The original and salient findings have included firstly a lower central sympathetic output to the periphery in women than in men, brought about by a greater baroreceptor reflex inhibitory effect by arterial blood pressure. Secondly, the vasoconstrictor effect of the peroneal sympathetic nerve activity was attenuated in women as compared to men, and this occurred both at resting conditions and during sympathetic activation.

Regarding group characteristics of men and women, some of our findings have already been previously reported. Thus, women have been found to be shorter [START_REF] Smulyan | Comparative effects of aging in men and women on the properties of the arterial tree[END_REF][START_REF] Ng | Age and gender influence muscle sympathetic nerve activity at rest in healthy humans[END_REF][START_REF] Tank | Baroreflex regulation of heart rate and sympathetic vasomotor tone in women and men[END_REF][START_REF] Fu | Effects of gender and hypovolemia on sympathetic neural responses to orthostatic stress[END_REF][START_REF] Hayward | Gender-related differences in the central arterial pressure waveform[END_REF][START_REF] Convertino | Gender differences in autonomic functions associated with blood pressure regulation[END_REF][START_REF] Evans | Gender differences in autonomic cardiovascular regulation: spectral, hormonal, and hemodynamic indexes[END_REF], to have a lower body weight [START_REF] Ng | Age and gender influence muscle sympathetic nerve activity at rest in healthy humans[END_REF][START_REF] Tank | Baroreflex regulation of heart rate and sympathetic vasomotor tone in women and men[END_REF][START_REF] Fu | Effects of gender and hypovolemia on sympathetic neural responses to orthostatic stress[END_REF][START_REF] Hayward | Gender-related differences in the central arterial pressure waveform[END_REF][START_REF] Convertino | Gender differences in autonomic functions associated with blood pressure regulation[END_REF][START_REF] Ettinger | Influences of gender on sympathetic nerve responses to static exercise[END_REF][START_REF] Barnett | Effects of age and gender on autonomic control of blood pressure dynamics[END_REF][START_REF] Christou | Women have lower tonic autonomic support of arterial blood pressure and less effective baroreflex buffering than men[END_REF], a slightly lower arterial blood pressure [START_REF] Wiinberg | 24-h ambulatory blood pressure in 352 normal Danish subjects, related to age and gender[END_REF][START_REF] Smulyan | Comparative effects of aging in men and women on the properties of the arterial tree[END_REF][START_REF] Convertino | Gender differences in autonomic functions associated with blood pressure regulation[END_REF][START_REF] Barnett | Effects of age and gender on autonomic control of blood pressure dynamics[END_REF][START_REF] Christou | Women have lower tonic autonomic support of arterial blood pressure and less effective baroreflex buffering than men[END_REF][START_REF] Huikuri | Sex-related differences in autonomic modulation of heart rate in middle-aged subjects[END_REF][START_REF] Peckerman | Effects of gender and age on the cardiac baroreceptor reflex in hypertension[END_REF] and higher heart rate [START_REF] Smulyan | Comparative effects of aging in men and women on the properties of the arterial tree[END_REF][START_REF] Hayward | Gender-related differences in the central arterial pressure waveform[END_REF][START_REF] Convertino | Gender differences in autonomic functions associated with blood pressure regulation[END_REF][START_REF] Christou | Women have lower tonic autonomic support of arterial blood pressure and less effective baroreflex buffering than men[END_REF][START_REF] Abdel-Rahman | Gender-related differences in the baroreceptor reflex control of heart rate in normotensive humans[END_REF] than men that were at least matched for age. These similarities could be considered to rule out the arguments that our subjects were not representative of other reported studies involving women and men.

Our previous findings and those of others have helped to design our study, in respect of avoiding confounding factors that can interfere with sympathetic nerve activity. This was achieved by matching individuals of the two groups. Subjects of both groups were Caucasians and were examined using the same protocol and under similar laboratory conditions whilst avoiding the influence of age, gender, dietary intake, general or regional obesity, large meal or visceral distension; these factors are known to affect sympathetic activity or its control [START_REF] Ng | Age and gender influence muscle sympathetic nerve activity at rest in healthy humans[END_REF][START_REF] Matsukawa | Gender difference in age-related changes in muscle sympathetic nerve activity in healthy subjects[END_REF][START_REF] Narkiewicz | Gender-selective interaction between aging, blood pressure, and sympathetic nerve activity[END_REF][START_REF] Sundlöf | Human muscle nerve sympathetic activity at rest. Relationship to blood pressure and age[END_REF][START_REF] Mary | The activity of single vasoconstrictor nerve units in hypertension[END_REF][START_REF] Sundlöf | The variability of muscle nerve sympathetic activity in resting recumbent man[END_REF]. Pre-and post-menopausal women were examined, as it has been shown that the onset of menopause does not affect the level of MSNA [START_REF] Narkiewicz | Gender-selective interaction between aging, blood pressure, and sympathetic nerve activity[END_REF]. The finding of slightly lower levels of arterial blood pressure in women was associated with a lower rather than higher sympathetic nerve activity than in men, a finding that is not consistent with an inhibitory effect through baroreceptor reflex control of sympathetic nerve activity. Also, the differences in sympathetic nerve activity between women and men were greater than the variability attending repeated measurements of this activity [START_REF] Greenwood | Single unit sympathetic discharge: quantitative assessment in human hypertensive disease[END_REF]. These considerations make it likely that the observed gender differences in sympathetic nerve activity were not solely caused by differences in the levels of arterial blood pressure or other factors known to affect the sympathetic output.

Our finding of a lower level of efferent sympathetic nerve activity in women than in men is similar to data found in the majority of other reports. For instance, six of nine studies have reported significantly lower sympathetic nerve activity in young and middle aged women than men [START_REF] Ng | Age and gender influence muscle sympathetic nerve activity at rest in healthy humans[END_REF][START_REF] Jones | Gender differences in muscle sympathetic nerve activity: effect of body fat distribution[END_REF][START_REF] Jones | Gender does not influence sympathetic neural reactivity to stress in healthy humans[END_REF][START_REF] Matsukawa | Gender difference in age-related changes in muscle sympathetic nerve activity in healthy subjects[END_REF][START_REF] Shoemaker | Gender affects sympathetic and hemodynamic response to postural stress[END_REF][START_REF] Narkiewicz | Gender-selective interaction between aging, blood pressure, and sympathetic nerve activity[END_REF]. In the remaining three reports, either women were found to have lower sympathetic activity than men when examined at the same arterial blood pressure [START_REF] Tank | Baroreflex regulation of heart rate and sympathetic vasomotor tone in women and men[END_REF] or the lower group average of sympathetic activity in women was not significantly different from that in men [START_REF] Ray | Effect of gender on vestibular sympathoexcitation[END_REF][START_REF] Fu | Effects of gender and hypovolemia on sympathetic neural responses to orthostatic stress[END_REF]. Of the three studies that examined older men and women, one found lower sympathetic activity in women than in men [START_REF] Ng | Age and gender influence muscle sympathetic nerve activity at rest in healthy humans[END_REF], while the other two [START_REF] Skarphedinsson | Sympathetic nerve traffic correlates with the release of nitric oxide in humans: implications for blood pressure control[END_REF][START_REF] Greenwood | Single unit sympathetic discharge: quantitative assessment in human hypertensive disease[END_REF] did not. Similarly, resting calf vascular resistance has been reported to be similar in women and men [START_REF] Ray | Effect of gender on vestibular sympathoexcitation[END_REF][START_REF] Dishman | Blood pressure and muscle sympathetic nerve activity during cold pressor stress: fitness and gender[END_REF]. Our study, which was primarily designed to test the possibility of gender-related differences, has established that nonobese and normal middle-aged women had lower resting efferent sympathetic nerve activity than men.

In both women and men, IHG and CPT caused significant increases in efferent sympathetic nerve activity and arterial blood pressure. This was not unexpected because the rise in arterial blood pressure that brings about a baroreceptor reflex inhibitory effect on efferent sympathetic activity and the rise of this activity in response to IHG and CPT are subject to complex interactions that include central effects [START_REF] Mark | Microneurographic studies of the mechanisms of sympathetic nerve responses to static exercise in humans[END_REF][START_REF] Kamiya | Static handgrip exercise modifies arterial baroreflex control of vascular sympathetic outflow in humans[END_REF][START_REF] Cui | Baroreflex modulation of muscle sympathetic nerve activity during cold pressor test in humans[END_REF][START_REF] Eckberg | Isometric exercise modifies autonomic baroreflex responses in humans[END_REF][START_REF] Scherrer | Arterial baroreflex buffering of sympathetic activation during exercise-induced elevations in arterial pressure[END_REF]. Previously reported data have not been consistent regarding a gender effect on the responses of arterial pressure and sympathetic activity to IHG [START_REF] Ng | Age and gender influence muscle sympathetic nerve activity at rest in healthy humans[END_REF][START_REF] Jones | Gender does not influence sympathetic neural reactivity to stress in healthy humans[END_REF][START_REF] Ettinger | Influences of gender on sympathetic nerve responses to static exercise[END_REF]. In the present study, the responses of arterial blood pressure to IHG were smaller in women, while the absolute increases of sympathetic nerve activity were similar to those in men leading to a greater percentage increase in women. In respect of CPT, the increases of arterial blood pressure and sympathetic activity in women were similar to those in men. Similar findings have previously been reported [START_REF] Ng | Age and gender influence muscle sympathetic nerve activity at rest in healthy humans[END_REF][START_REF] Jones | Gender does not influence sympathetic neural reactivity to stress in healthy humans[END_REF][START_REF] Shoemaker | Gender affects sympathetic and hemodynamic response to postural stress[END_REF][START_REF] Dishman | Blood pressure and muscle sympathetic nerve activity during cold pressor stress: fitness and gender[END_REF].

The mechanisms of the lower resting efferent sympathetic nerve activity in women were shown in this study to involve a greater baroreceptor reflex inhibitory control of this activity in women than in men. The BRS-sna/dbp in women was steeper than in men, and was accompanied by an impaired BRS-hp/sbp in women relative to men. The latter has previously been found using the Valsalva manoeuvre as in the present study [START_REF] Huikuri | Sex-related differences in autonomic modulation of heart rate in middle-aged subjects[END_REF], using neck suction [START_REF] Convertino | Gender differences in autonomic functions associated with blood pressure regulation[END_REF] as well as when changing arterial blood pressure levels by pharmacological agents [START_REF] Peckerman | Effects of gender and age on the cardiac baroreceptor reflex in hypertension[END_REF][START_REF] Abdel-Rahman | Gender-related differences in the baroreceptor reflex control of heart rate in normotensive humans[END_REF]. As such our method of examining baroreceptor reflex control has yielded similar results to those obtained using other techniques. In respect of the control of efferent sympathetic nerve activity, previously reported studies in animals and humans have indicated that females have a greater central and baroreceptor reflex inhibitory control of this activity than males [START_REF] Rio | Sex differences in catecholamine response to clonidine[END_REF][START_REF] Hinojosa-Laborde | Gender differences in sympathetic nervous system regulation[END_REF]. Our study has now established that women have a greater baroreceptor reflex inhibitory control of the sympathetic activity, and also found a smaller control of heart rate that is mainly mediated by withdrawal of vagal effects. These considerations are consistent with the findings in this study of a lower resting sympathetic nerve activity and higher heart rate in women than in men.

Another new finding of our study was the demonstration that the vasoconstrictor effect of the efferent sympathetic activity in women was attenuated relative to that in men. This was apparent in the relationship between the sympathetic activity and either CVR or CBF that was found only in men; greater sympathetic activity was associated with greater CVR and lower CBF in men and not in women. In addition, women had a smaller increase of CVR than men during augmentation of efferent sympathetic activity by CPT and IHG. Previously reported studies, mainly in animals, have found an attenuated vascular response to sympathetic stimulation in females [START_REF] Hinojosa-Laborde | Gender differences in sympathetic nervous system regulation[END_REF]. The present investigation has now established such gender-related difference in humans.

In the present study we quantified the central output of sympathetic nerve activity to the periphery, and as such this could differ from that destined to supply visceral organs [START_REF] Esler | The sympathetic system and hypertension[END_REF]. However, in normal subjects a correlation has been reported between MSNA and the sympathetic drive to the heart and the kidney as assessed by norepinephrine spillover rate [START_REF] Wallin | Simultaneous measurements of cardiac noradrenaline spillover and sympathetic outflow to skeletal muscle in humans[END_REF][START_REF] Wallin | Renal noradrenaline spillover correlates with muscle sympathetic activity in humans[END_REF]. Furthermore, it has been reported that men have a predominance of sympathetic effects on the heart interval using spectral analysis of the heart rate [START_REF] Tank | Baroreflex regulation of heart rate and sympathetic vasomotor tone in women and men[END_REF][START_REF] Barnett | Effects of age and gender on autonomic control of blood pressure dynamics[END_REF][START_REF] Huikuri | Sex-related differences in autonomic modulation of heart rate in middle-aged subjects[END_REF][START_REF] Ramaekers | Heart rate variability and heart rate in healthy volunteers. Is the female autonomic nervous system cardioprotective?[END_REF][START_REF] Kuo | Effect of aging on gender differences in neural control of heart rate[END_REF], and higher circulating catecholamine levels than women [START_REF] Jones | Gender does not influence sympathetic neural reactivity to stress in healthy humans[END_REF][START_REF] Evans | Gender differences in autonomic cardiovascular regulation: spectral, hormonal, and hemodynamic indexes[END_REF][START_REF] Christou | Women have lower tonic autonomic support of arterial blood pressure and less effective baroreflex buffering than men[END_REF].

These reported findings are consistent with our results of an existing difference in the sympathetic drive between women and men.

Our findings raise clinical implications regarding gender differences in respect of conditions related to sympathetic activation. For instance, the level of arterial blood pressure and the incidence of cardiovascular disease, including hypertension, have been reported to be lower in middle-aged women than in men of similar age [START_REF] Wiinberg | 24-h ambulatory blood pressure in 352 normal Danish subjects, related to age and gender[END_REF][START_REF] Burt | Prevalence of hypertension in the US adult population. Results from the Third National Health and Nutrition Examination Survey, 1988-1991[END_REF]. Indeed, a lower prevalence of cardiovascular disease has been documented in women than in men [START_REF] Lerner | Patterns of coronary heart disease morbidity and mortality in the sexes: a 26-year follow-up of the Framingham population[END_REF][START_REF] Mosca | Cardiovascular disease in women. A statement for healthcare professionals from the American Heart Association[END_REF][START_REF] Reckelhoff | Gender differences in the regulation of blood pressure[END_REF]. These reports could be explained on the basis of our findings of a lower level of sympathetic output and its attenuated vasoconstrictor effect.

In conclusion our study has demonstrated that women have a lower central sympathetic nerve activity to the periphery, the mechanism of which involved a greater baroreceptor reflex inhibitory effect on this activity in women than in men. The study also demonstrated an attenuated vasoconstrictor effect of peroneal efferent sympathetic nerve activity in women than in men. These findings could have implications regarding the lower cardiovascular events observed in women than in men.

Figure 1

 1 Figure 1.

Figure 4

 4 Figure 4.

Figure 1 .

 1 Figure 1. Levels of m-MSNA (multi-unit muscle sympathetic nerve activity) and s-MSNA (single unit muscle sympathetic nerve activity) expressed per 100 cardiac beats (top) and per minute (bottom) for men and women. P values refer to unpaired t tests when significant. The height of the column denotes mean of groups and the bars SEM. Women have lower sympathetic activity than men.

Figure 2 .

 2 Figure 2. Baroreceptor reflex sensitivity for men and women. BRS-hp/sbp refers to the control of the heart period (top) and BRS-sna/dbp to the control of efferent sympathetic nerve activity (bottom). P values refer to unpaired t tests, the column denotes mean of groups and the bars SEM. Women have lower BRS-hp/sbp and greater BRS-sna/dbp than men.

Figure 3 .

 3 Figure 3. The relationship of MSNA (bursts/100beats) to A) CVR and B) CBF in men (left) and women (right). MSNA was positively correlated to CVR and negatively correlated to CBF on men with no such significant correlations in women.

Figure 4 .

 4 Figure 4. Absolute responses of heart rate and mean arterial blood pressure (top), and of m-MSNA, s-MSNA and calf vascular resistance (bottom) to cold pressor test for men and women. P values refer to unpaired t tests. The height of the column denotes mean of groups and the bars SEM. Women have an attenuated increase in CVR in response to cold pressor test relative to men.

Figure 5 .

 5 Figure 5. Absolute responses of heart rate and mean arterial blood pressure (top), and of m-MSNA, s-MSNA and calf vascular resistance (bottom) to isometric handgrip test for men and women. P values refer to unpaired t tests. The height of the column denotes mean of groups and the bars SEM. Women have an attenuated increase in calf vascular resistance in response to isometric handgrip test relative to men.

Table 1 . Data of the two groups of 34 women and 34 men.

 1

	Variable	Women	Men	Significance P
	Age (years)	43±2.2	45±2.6	Ns
	Body weight (kg)	69±1.7	83±1.6	<0.0001
	Height (cm)	165±1.0	179±1.3	<0.0001
	BMI (kg/m 2 )	25±0.6	26±0.5	Ns
	Waist circumference (cm)	84.9±1.8	87.2±2.0	Ns
	Heart rate (beats/minute)	64±1.2	61±1.6	Ns
	Mean ABP (mmHg)	93±1.3	96±1.0	0.04
	Systolic ABP (mmHg)	123±1.8	128±1.6	0.04
	Diastolic ABP (mmHg)	78±1.0	80±1.0	Ns
	s-MSNA (impulses/100b)	39±3.6	58±3.8	0.0003
	s-MSNA (impulses/min)	24±2.0	34±2.3	0.0007
	m-MSNA bursts (bursts/100b)	33±3.1	50±3.8	0.0007
	m-MSNA bursts (bursts/min)	21±1.8	29±2.3	0.0026
	CBF (ml.100ml. -1 min. -1 )	2.3±0.08	2.4±0.13	Ns
	CVR (units)	41±2.0	44±2.4	Ns
	BRS-hp/sbp (ms/mmHg)	5.3±0.40	7.2±0.55	0.0035
	Data as mean ± SEM. BMI (body mass index). ABP (arterial blood pressure). m-MSNA
	(multi-unit muscle sympathetic nerve activity) and s-MSNA (single unit muscle
	sympathetic nerve activity) expressed per 100 cardiac beats (100b) and per minute
	(min). P values refer to two tailed unpaired t tests, ns as p ≥ 0.05

Acknowledgements

The authors thank Mr. Jeff Bannister and Mrs. Julie Corrigan for technical assistance, and the British Heart Foundation for sponsorship (Grant No: FS04/085).

This work was sponsored by the British Heart Foundation (Grant No: FS/04/085)

The authors declare no conflicting interests


Dr Christian J F Holubarsch
email: c.holubarsch@median-bk.de

Martin Rohrbach

Matthias Karrasch

Erich Boehm

Lech Polonski

Piotr Ponikowski

Siegfried Rhein

A double

Keywords: drugs, fatty acids, glucose, heart failure, metabolism Clinical Science Immediate

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction:

Congestive Heart Failure (CHF) is a major public health problem afflicting up to 3% of the population in western countries and is associated with high morbidity and mortality rates [START_REF] Ho | The epidemiology of heart failure: the Framingham study[END_REF][START_REF] Mcmurray | An evaluation of the cost of heart failure to the National Health Service in the UK[END_REF][START_REF] Andrews | Clinical and economic factors in the treatment of congestive heart failure[END_REF] Heart failure can be viewed upon as the end-stage of various forms of heart disease [START_REF] Mostered | Prevalence of heart failure and left ventricular dysfunction in the general population; The Rotterdam Study[END_REF]. The prognosis for patients with heart failure is poor and is in fact even worse than the survival chances in patients suffering from various malignancies [START_REF] Murray | AD-Global mortality, disability and the contribution of risk factors: global burden of disease study[END_REF] Various surveys have shown that medical management of CHF is characterized by polypharmacy and by the underuse of recommended medications [START_REF] Cohen-Solal | for the myocardial and heart failure working group of the French Society of Cardiology, the National College of General Hospital Cardiologists and the French Geriatrics Society. A national survey of heart failure in French hospitals[END_REF][START_REF] Taubert | Clinical profile and management of heart failure: rural community hospital vs metropolitan heart center[END_REF].

The conventional medical therapy for CHF has been extensively outlined [START_REF] Force | Guidelines for the evaluation and management of heart failure. Report of the American College of Cardiology/American Heart Association Task Force on practice guidelines (committee on evaluation and management heart failure)[END_REF]. The following classes of agents are used: Diuretics are used to control the symptoms of volume overload. When these symptoms are absent, diuretics, in form of loop diuretics, have no proven benefit [START_REF] Packer | for the RADIANCE Study. Withdrawal of digoxin from patients with chronic heart failure treated with angiotensin-converting (enzyme inhibitors)[END_REF]. However, spironolactone [START_REF] Pitt | RALES-study[END_REF] and eplerenone [START_REF] Pitt | Eplerenone post-AMI heart failure efficacy and survival study: primary results of the EPHESUS-trial[END_REF] have shown a clear benefit for heart failure patients regarding survival. The second class of agents useful in CHF includes digitalis glycosides, which have been shown to improve symptoms but not to prolong life [START_REF] Digitals | The effect of digoxin on mortality and morbidity in patients with heart failure[END_REF]. The next class of agents used in the pharmacogical therapy of CHF is the vasodilators. The angiotensinconverting enzyme (ACE) inhibitors have the most beneficial effect on mortality and are the most commonly used [START_REF] Cohn | A comparison of enalapril with hydralazinisosorbide dinitrate in the treatment of chronic congestive heart failure[END_REF][START_REF] The | Effect of enalapril on survival in patients with reduced left ventricular ejection fractions and congestive heart failure[END_REF][START_REF] Pfeffer | Effect of captopril on mortality and morbidity in patients with left ventricular dysfunction after myocardial infarction: results of the survival and ventricular enlargement trialthe SAVE investigators[END_REF][START_REF]Effect of ramipril on mortality and morbidity of survivors of acute myocardial infarction with clinical evidence of heart failure[END_REF]. The most recent class to be recommended for heart failure patients is the beta-blockers, which provide a number of beneficial effect: Decrease in heart rate, decrease in overall work load, reduction in oxygen consumption, prolongation of diastolic myocardial perfusion, protection against toxic effects of catecholamines. These effects may work in common and thereby increase survival [START_REF] Packer | The effect of carvedilol on morbidity and mortality in patients with chronic heart failure. U.S. Carvedilol Heart Failure Study Group[END_REF][START_REF] Packer | Effect of Carvedilol on Survival in Severe Chronic Heart Failure[END_REF][START_REF]The Cardiac Insufficiency Bisoprolol Study II (CIBIS-II): a randomized trial[END_REF].

Despite the use of recent therapeutic regimens, many patients remain symptomatic and are at high risk of cardiac death. There remains a need to develop new treatments for heart failure which extend survival, reduce symptoms and provide patients with an improved capacity to conduct daily activities.

Etomoxir is an inhibitor of mitochondrial carnitine-palmitoyl-transferase 1 (CPT1), developed originally for the treatment of diabetes mellitus [START_REF] Group | Effect of metoprolol CR/XL in chronic heart failure: Metoprolol CR/XL Randomised Intervention Trial in Congestive Heart Failure (MERIT-HF)[END_REF]. This inhibition of mitochondrial CPT1 is common to a number of oxirane carboxylic acid derivatives and is both irreversible and stereospecific. Etomoxir effectively blocks long-chain fatty acid oxidation in heart, skeletal muscle and liver and thus switches the metabolism to glucose oxidation. Such a switch has been shown to induce expression of sarcoplasmic reticulum calcium ATPase and redistribution of myosin isoenzymes in experimental animal studies -alterations which may be beneficial in chronic heart failure [START_REF] Wolf | Aryl-substituted 2-oxirane carboxylic acids: a new group of antidiabetic drugs[END_REF][START_REF] Vetter | CPT-1 inhibition by etomoxir has a chamberrelated action on cardiac sarcoplasmic reticulum and isomyosins[END_REF][START_REF] Vetter | Differential influences of carnitine palmitoyltransferase-1 inhibition and hyperthyroidism on cardiac growth and sacroplasmi reticulum phosphorylation[END_REF][START_REF] Rupp | Dietary medium-chain triglycerides can prevent changes in myosin and SR due to CPT-1 inhibition by etomoxir[END_REF][START_REF] Rupp | Modification of subcellular organelles in pressure-overloaded heart by etomoxir , a carnitine palmitoyltransferase 1 inhibitor[END_REF].

In a first clinical trial with etomoxir in patients with chronic congestive heart failure, clinical improvement has been reported for 80 mg etomoxir per day [START_REF] Zarain-Herzberg | Modification of sarcoplasmic reticulum gene expression in pressure overload cardiac hypertrophy by etomoxir[END_REF].

The purpose of this study was to reconfirm the efficacy of 80 mg etomoxir per day in A total of 230 (66%) patients had ischemic heart disease of which 80 (35%) had current angina and 182 (79%) reported at least one previous myocardial infarction. 189 (57%) patients had hypertension, 84 (24%) had dilated cardiomyopathy, 21 (6%) had valvular heart disease, and 14 (4%) had other etiologies. 59 (17%) of the patients had diabetes mellitus type 2.

All patients gave written consent. The protocol was reviewed and approved by competent Ethic Committees

Study protocol: A double-blind study design was used. Etomoxir was provided in soft gelatine capsules containing 40mg compound dissolved in a mixture of medium chain triglycerides derived from coconut and palm oil. The daily dose for one patient was packed into blister strips, each strip containing two capsules for each seven days; as one of the following three forms: 80mg etomoxir (two 40mg capsules), 40mg etomoxir (one verum and one placebo capsule), or placebo (two placebo capsules).

All patients were given two capsules once daily prior to their evening meal.

Examination: Before randomization, patients completed a baseline six-minutecorridor-walk-test, two quality of life questionnaires (Minnesota 'Living with heart failure' and EUROQOL 'EQ-5D') and a 24 hour Holter ECG. The treatment phase was thought to contain visits at 3, 6, 12, 18 and 24 weeks following randomization.

The visits consisted of physical examination, 12-lead ECG, urinalysis, bicycle protocol and a six-minute-corridor-walk-test. The patients underwent laboratory examinations including electrolytes, liver and renal function and hematology profile with platelet count at 3, 6, 18 and 28 week visits and biochemistry and hematology at 12 and 24 week visits. At 3 and 24 week visits 24-hour Holter monitoring was performed.

Exercise testing: Bicycle protocol and the six-minute-corridor-walk-test were performed at baseline, 12 and 24 week visit (bicycle protocol was optional at 3, 6 and 18 week visits). At baseline at least two exercise tests were postulated with exercisetimes within ± 20%.

The bicycle protocol consisted of two minutes stages with an initial work load of 25 watts, increasing by 25 watts each successive stage.

All bicycle tests for a particular patient were scheduled for the same time of day throughout the study. The following test measurements were determined: date and time of start test; total exercise time; ECG at the end of every minute and at 1, 2, and 5 minutes post exercise; systolic and diastolic blood pressure; heart rate at the end of each two minute stage, at the end of exercise and at 1, 3, and 5 minutes post exercise; Borg score (end of each stage; at end of exercise); reason for stopping if needed.

The six minute corridor walk test was thought to be a simple and objective measure of submaximal exercise capacity. On all occasions this test was performed during a visit where the bicycle test has previously been performed.

Echocardiography: Echocardiographical measurements were made as follows: the left ventricular ejection-fraction LVEF, left ventricular end-diastolic diameter LVEDD, fractional shortening and left ventricular wall thickness at the septum and the posterior wall. Echocardiograms were performed at baseline and at the 24-week-endpoint.

Holter monitoring: Twenty-four hour Holter monitoring was performed at 3, 12 and 24 week visits. All evidences of torsade de pointes and ventricular fibrillations, asystoles, runs of ventricular tachycardia, AF, flutter or supraventricular tachycardia, number of ectopic beats, number of ectopic couplets and mean heart rate were printed out and validated by a cardiologist.

Sample size calculation:

The proposed recruitment of 120 patients per treatment group (that is, a total of 360 patients who successfully complete the screening period and are eligible for randomisation) is motivated by the following considerations:

• An average baseline total exercise time for the bicycle protocol of 420 seconds (seven minutes).

• An anticipated increase over baseline by week 24 of 45 seconds for placebo, 67.5 seconds for 40 mg etomoxir and 90 seconds for 80 mg etomoxir. Some placebo effect is anticipated as the baseline value will be obtained prior to the commencement of study medication.

• A within-patient standard deviation for change from baseline with respect to the primary endpoint of 90 seconds. This figure is consistent with the requirement that prior to randomisation each patient must achieve two exercise times within ± 20%.

Clinical Science Immediate Publication. Published on 23 Feb 2007 as manuscript CS20060307

• The direct comparison of 80 mg etomoxir with placebo, to be investigated using a two sided parametric test at the experimentwise 5% significance level should have at least 90% power.

Assuming equal numbers of patients per group, it can be shown via simulation that the ANOVA F test for the equality of the three treatment means has 90% power if a minimum of 103 patients are recruited to each group and the underlying group means are as described. This is a "worst-case" scenario and it can be shown that in this case the comparison of 80 mg etomoxir with placebo will also be significant with a power of at least 90%

Statistical analysis:

Comparability of treatment groups at baseline has been assessed and summarized using descriptive statistics. Hypothesis testing to determine differences between treatments were two-sided, with differences being declared statistically significant if the observed significance level is less than 0.05. The two pairwise comparisons of 80mg etomoxir with placebo and 40mg etomoxir with placebo with respect to the primary endpoint constituted the only confirmatory analysis and the type I error for this analysis was controlled to be 0.05 by adjusting the statistics using the method of Dunnett. All other analyses were secondary and were reported without adjustment of significance levels.

Data collection and the statistical analysis were conducted on a collaborative basis by the Study Statistician and the Statistic Department of Nottingham Clinical

Research Limited who jointly prepared the Statistical Analysis Plan.

Results:

Characteristics of the treatment groups.

A total of 375 patients had some data entered into the study data base with 347 patients randomised with 121 receiving placebo, 118 patients receiving 40 mg and 108 receiving 80 mg etomoxir. Table 1 gives details on entry and completion for randomised patients for each country. Therefore at the end of the prematurely stopped study, 52 patients had completed the study, 21 in the placebo group, 17 in the 40 mg etomoxir and 14 in the 80 mg etomoxir group respectively.

The treatment groups were balanced with respect to age, gender, smoking history and race. Table 2 summarizes demographics information about treatment groups.

A total of 209/347 (60%) patients were graded NYHA grade II at screening. The corresponding number of patients graded NYHA III was 137 (40%), see also Table 2.

Concommitant Medication:

81% of the patients were on ACE-inhibitors or angiotensin-receptor-blockers, 52% on ß-blockers, 23% on spironolactone, 7% on amiodarone, 32% on nitrates, and 24% digitalis-preparations.

Bicycle Exercise testing and echocardiographical data:

A total of 234 patients were included in the randomised set for the principal measure of efficacy of the change from baseline in total exercise time (sec) at week 24 endpoint.

There was a clear tendency regarding increases in bicycle exercise testing (Figure 1;

Table 3). The mean increases in exercise times were 3.3 sec, 10.2 sec and 19.4 sec for the placebo, etomoxir 40 mg and etomoxir 80 mg groups, respectively (Table 3).

However, none of these were statistically significant. The number of patients that reached the 24 week endpoint and thus could be included in the pairwise comparisons were n = 21 in the placebo group, n = 16 in 40 mg and n = 14 in the 80 mg etomoxir groups respectively (Table 3). This may be due to the premature study end resulting in a too small number of patients having finished the study. There were no statistically significant differences between any of the three pairwise comparisons for the change from baseline in total distance covered in the six-minute-corridor-walk test at week 12 endpoint and week 24 endpoint (table 4 A). Also, no statistically significant differences were found for LVEF (table 4 B), LVEDD, fractional shorting and left ventricular wall thickness at week 12 and week 24 endpoint.

Unfortunately, there is no data available from the 24 hours Holter monitoring.

Cardiac deaths and serious adverse events:

Six patients died during the study. One patient died during the run-in phase. Five patients died during the randomisation phase, three in the placebo group all of whom had sudden cardiac death, one in the etomoxir 40 mg group who died due to cardiogenic shock, and one patient in the 80 mg etomoxir group who died due to myocardial infarction.

Serious adverse events are listed in table 5.There were four patients with elevations of liver enzymes all of them were either in the 40 mg etomoxir or in the 80 mg etomoxir group ( see table 6).

Using the Minnesota questionnaire:

The pairwise comparison between 40 mg etomoxir and placebo was statistically significant (p<0.05) in favour of 40 mg etomoxir for the following questions and timepoints:

Making him/her sit or lie down to rest during the day at week 24 endpoint, making going places away from home difficult at week 12 endpoint, and making them stay in a hospital at week 24 endpoint.

The pairwise comparison between 80 mg etomoxir and placebo was statistically significant (p<0.05) in favour of 80 mg etomoxir for making them stay in a hospital at week 24 endpoint. The former two alterations may be judged to be beneficial, in as far as they compensate for increased wall stress. Due to the vasodilating, diuretic, and angiotensin-converting-enzym blocking properties of ANP and BNP, these peptides ameloriate increased workload, and salt and water retention [START_REF] Schmidt-Schweda | First Clinical trial with etomoxir in patients with chronic congestive heart failure[END_REF][START_REF] Takahashi | Expression of A-, B-, and Ctype natriuretic peptide genes in failing and developing human ventricles, Correlation with expression of the Ca 2+ -ATPase gene[END_REF][START_REF] Morita | Increased plasma levels of brain natriuretic peptide in patients with acute myocardial infarction[END_REF][START_REF] Hama | Rapid ventricular induction of brain natriuretic peptide gene expression in experimental acute myocardial infarction[END_REF][START_REF] Wiese | Gene Expression of Brain Natriuretic Peptide in Isolated Atrial and Ventricular Human Myocardium. Influence of Angiotensin II and Fiber Length[END_REF]. The shift of myosin heavy chains from αα to ββ-dimers leads to slower actomyosin cross-bridge cycling rates and thereby improved economy of force generation as shown in animal experimental studies [START_REF] Nakao | Roles of natriuretic peptides in heart failure[END_REF][START_REF] Alpert | Increased myothermal economy of isometric force generation in compensated cardiac hypertrophy induced by pulmonary artery constrictioin in rabbit. A characterization of heat liberation in normal and hypertrophied right ventricular papillary muscle[END_REF]34,35,36]. Such a shift may also play a role in human myocardium [37,38,39,40] which may also be interpreted to be beneficial because of more economical contraction.

In contrast, a decrease of SERCA-2 on the one hand and an increase in sodiumcalcium exchanger on the other may be associated with decreased calcium activity, and thereby impairment of diastolic and systolic function [37,38,39,40]. Therefore, to counteract the latter two alterations may be a therapeutic goal [41,42,43].

Experimental animal studies with etomoxir

Etomoxir has originally been developed for treatment of diabetes mellitus [START_REF] Group | Effect of metoprolol CR/XL in chronic heart failure: Metoprolol CR/XL Randomised Intervention Trial in Congestive Heart Failure (MERIT-HF)[END_REF].

Etomoxir blocks mitrochondrial CPT-1 thereby switching the metabolism from fattyacid-oxidation to glucose oxidation [START_REF] Group | Effect of metoprolol CR/XL in chronic heart failure: Metoprolol CR/XL Randomised Intervention Trial in Congestive Heart Failure (MERIT-HF)[END_REF]. This metabolic switch may have some advantage regarding energy expenditure under ischemic conditions [44], but --10probably more importantly -influences gene expression of SERCA-2 and myosin isoenzymes [START_REF] Wolf | Aryl-substituted 2-oxirane carboxylic acids: a new group of antidiabetic drugs[END_REF][START_REF] Vetter | CPT-1 inhibition by etomoxir has a chamberrelated action on cardiac sarcoplasmic reticulum and isomyosins[END_REF][START_REF] Vetter | Differential influences of carnitine palmitoyltransferase-1 inhibition and hyperthyroidism on cardiac growth and sacroplasmi reticulum phosphorylation[END_REF] thereby counteracting the molecular mechanisms taking place in congestive heart failure [START_REF] Rupp | Dietary medium-chain triglycerides can prevent changes in myosin and SR due to CPT-1 inhibition by etomoxir[END_REF][START_REF] Rupp | Modification of subcellular organelles in pressure-overloaded heart by etomoxir , a carnitine palmitoyltransferase 1 inhibitor[END_REF]. Therefore, it was an attractive pharmacological approach to test etomoxir in patients suffering from congestive heart failure.

Clinical Studies with Etomoxir:

In a first, non-placebo controlled study in a small number of patients, this pharmacological approach was judged to be positive and safe, when etomoxir was given over a period of three months [START_REF] Zarain-Herzberg | Modification of sarcoplasmic reticulum gene expression in pressure overload cardiac hypertrophy by etomoxir[END_REF]. In this earlier study, we demonstrated a significant increase in maximum workload, which was associated with an increased ejection fraction and improved hemodynamic parameters. On this basis, a placebocontrolled much larger trial, the ERGO-Study, was planned and conducted with two etomoxir dosages (40 mg and 80 mg ) over a period of six months.

In contrast to our earlier study, the present ERGO-Study did not show significant improvement in exercise time or in echocardiographic parameters. The reason for these discrepancies may be threefold:

(1) In the earlier study, nine out of ten patients suffered from dilated cardiomyopathy, whereas in the present study 66% of the patients had ischemic heart disease. It may well be that etomoxir is more effective in patients with cardiomyopathy than in those with ischemic heart disease.

(2) The degree of the disease was more advanced in the earlier study as indicated by a mean ejection fraction of only 21.5%, whereas in the present study mean ejection fraction was 31%. Therefore, the extent of possible improvement may be different between the two studies.

(3) As shown in Figure 1 and Table 3, there was a tendency for an increase in exercise-time of about 20 seconds in the 80 etomoxir group. With a greater number of patients instead of only fourteen in the pairwise comparison this -11beneficial effect might become significant. Unfortunately, due to the hepatotoxic effects of etomoxir, the ERGO-Study had to be stopped prematurely (see below).

(4) Beside the fact that subjects had a tendency (NS) to exercise more, some issues of the Minnesota questionnaire were significantly different in favour of etomoxir.

Hepatotoxicity of etomoxir and premature end of the study:

The ERGO-Study was prematurely stopped because in four patients liver enzymes were found to be elevated. In the study protocol, blood tests including liver enzymes were planned at 3, 6, 12, 18 and 24 weeks following randomisation. In former studies, increases in transaminase activity were reported, however these occurred early, were mild (increase by a factor for two), and of transient character [START_REF] Group | Effect of metoprolol CR/XL in chronic heart failure: Metoprolol CR/XL Randomised Intervention Trial in Congestive Heart Failure (MERIT-HF)[END_REF]. The following arguments were the most important for the responsible committee to stop the continuation of the study:

(1) There was a clear relationship between increases in ALT and AST and study medication: None of the four patients with increased liver enzymes was in the placebo-group, one was in the 40 mg and three were in the 80 mg etomoxir group.

Furthermore, in all patients ALT and AST returned to normal values after cessation of etomoxir.

(2) A number of four patients with liver problems seems low at the first glance.

However at the time the study was stopped, only 15% of the study population had already safely completed the study.

(3) The increase in ALT and AST occurred relatively late, i.e., at week six after randomisation. Therefore many randomised patients of the study were still at risk.

(4) The increase of ALT and AST were unexpectedly high and not at all transient.

Therefore, these changes did not indicate an adaptation of a liver cell but rather cell toxicity and cell death.

(5) Under these conditions, etomoxir was judged to be no candidate for further development for treatment of heart failure patients because of too severe adverse events.

Clinical Science Immediate Publication. Published on 23 Feb 2007 as manuscript CS20060307 [START_REF] Cohen-Solal | for the myocardial and heart failure working group of the French Society of Cardiology, the National College of General Hospital Cardiologists and the French Geriatrics Society. A national survey of heart failure in French hospitals[END_REF] All together the risk-to-benefit-ratio did not justify the continuation of the study for the individuals being at risk. However, despite of premature stop of the study, there was a tendency to increased exercise time, the primary endpoint of the study. Therefore, before rejecting the hypothesis that fatty-acid-oxidation-inhibition might be beneficial in congestive heart failure, similar studies have to be performed using different fatty-acid-oxidationinhibitors targeting CPT-1 and other enzymes of the metabolic pathway.

Alteration of cardiac energy metabolism, i.e., switch from fatty acids to glucose oxidation, may be achieved by alternative medical approaches:

(1) Trimetazadine has a similar metabolic effect like etomoxir by inhibiting mitochondrial long-chain 3-ketoacyl coenzyme A thiolase [46]. Cardioprotective effects of this compound have been described during coronary artery graft surgery [47], but no data are available regarding its effect in heart failure patients.

(2) Glucagon-like peptide-1 (GLP-1) or a similiarly active substance -exenatideare known to stimulate insulin release -dependent on actual glucose concentrations. Such a medical intervention may not only act to control serum glucose concentration, but also alter cardiac metabolism favourably.

(3) So-called insulin-sensitizers like pioglitazone and rosiglitazone are currently used as anti-diabetics. Due to their cellular effects on glucose-availability, a positive effect on cardiac energy metabolism may also be present in heart failure patients, which needs to be studied.

In the light of future alternatives, the strategy to alter cardiac metabolic pathways by compounds other than etomoxir remains still an attractive and challenging pharmacological approach. The numbers in parenthesis indicate the numbers of patients from which data were available for analysis. Normal range for ATL is up to 35 U/l and for AST is up to 45 U/l.

Values for gamma-GT, Bilirubin, and Alcaline phosphatase remained in the normal range. After withdrawal of etomoxir, ALT and AST normalized gradually.

  a double-blind setting and to study the risk/benefit ratio of 40 mg and 80mg etomoxir in comparison with placebo based on clinical findings, exercise testing, echocardiography and Holter monitoring in patients with moderate congestive heart failure (CHF NYHA class II-III). Methods: Rationale: The hypothesis to be tested in the ERGO-Study was an increase in cardiopulmonary capacity by 10% as induced by etomoxir at a dosage of 40 or 80 mg compared to placebo during a period of three or six months. Patients: Investigations were performed in Poland, Germany and Italy. 347 patients were randomised, 193 patients in Poland, 127 patients in Germany, and 27 patients in Italy. Age of patients was between 21 and 84 years (mean 66.6 years). Patients were suffering from moderate congestive heart failure (NYHA II -III).

  Molecular changes of the myocardium in congestive heart failure Left ventricular systolic dysfunction and chronic congestive heart failure are known to be associated not only with a quantitative increase in muscle mass, but also with subtle qualitative changes in gene expression and subsequent district functional consequences of the myocardium. At least four major phenotypic changes have been reported: (1) Increased production and release of natriuretic peptid type A and type B (ANP and BNP), (2) a shift in the myosin heavy chains from αα to ββ dimers, (3) upregulation of the gene encoding the sodium-calcium exchanger, and (4) dowregulation of SERCA-2.

  the control value before randomisation, t 1 is the blood control after 3 weeks and t 2 after 6 weeks of treatment N means within the normal range.

Clinical Science Immediate Publication. Published on 23 Feb 2007 as manuscript CS20060307

Clinical Science Immediate Publication. Published on 23 Feb 2007 as manuscript CS20060307 34.

  Holubarsch, Ch., Litten, R.Z., Mulieri, L.A., and Alpert, N.R. (1995) Energetic changes of myocardium as an adaptation to chronic hemodynamic overload and thyroid gland activity. Bas. Res. Cardiol. 80, 582-593 35. Holubarsch, Ch., Goulette, R. P., Litten, R.Z. et al. (1985) The economy of isometric force development, myosin isoenzyme pattern and myofibrillar ATPase activity in normal and hypothyroid rat myocardium. Circ. Res. 56, 78-86 36. Hasenfuß, G., Mulieri, L.A., Blanchard, E.M. et al. (1991) Energetics of isometric force development in control and volume-overload human myocardium.Comparison with animal species. Circ. Res. 68, 836-846 37. Berman, M.R., Lord, C.C., and Maughan, D.W. (1988) Force transient course in heart muscle with high and low V 1 to V 2 myosin isoenzyme ratio J. Mol. Cell. Cardiol. 20, 679-687 38. Hasenfuss, G., Reinecke, S., Studer, R. et al. (1994) Relation between myocardial function and expression of sarcoplasmic reculum Ca 2+ -ATPase in failing and nonfailing human myocardium. Circ. Res. 75, 434-442 39. Holubarsch, Ch., Schneider, R., Pieske, B. et al. (1995) Positive and negative inotropic effects of DL-Sotalol and D-Sotalol in failing and nonfailing human myocardium under physiological experimental conditions. Circulation 92, 2904-2910 40. Pieske, B., Kretschmann, B., Meyer, M. et al. (1995) Alterations in intracellular calcium handling associated with the inverse force-frequency relation in human dilated cardiomyopathy. Circulation 92, 1169-1178 41. Feldmann, A.M., Weinberg, E.O., Rax, P. E., and Lorell, B.H. (1993) Selective changes in cardiac gene expression during compensated hypertrophy and the transition to cardiac decompensation in rats with chronic heart aortic banding.

	Table1. Patients entry and completion by country
	Country	Placebo Entered Completed Entered Completed Entered Completed Etomoxir 40mg Etomoxir 80mg
	Germany 47	21	41	17	39	14
	Italy	8	-	10	-	9	-
	Poland	66	-	67	-	60	-
	Overall	121	21	118	17	108	14
	Circ. Res. 73, 184-192
	42. Hajjar, R.J., Kang, J.X., Gwathmey,J.K., and Rosenzweig, A. (1996)
	Physiological effects of adenoviral gene transfer of sarcoplasmic reticulum
	calcium ATPase in isolated rat myocytes. Circulation 94, (Suppl.I), 89
	43. Hajjar, R.J., Kang, J.X., Gwathmey, J.K., and Rosenzweig, A. (1997)
	Physiological effects of adenoviral gene transfer of sarcoplasmic reticulum
	calcium ATPase in isolated rat myocytes. Circulation 95, 423-429
	44. Meyer, M., and Dillmann, W.H. (1998) Sarcplasmic reticulum Ca 2+ -ATPase
	overexpression by adenovirus mediated gene transfer and in transgenic
	animals. Cardiovasc. Res. 37, 360-366
	45. Lopaschuk, G.D., Spaffort, M.A., Davies, N.J., and Wall, S.R. (1990) Glucose
	and palmitate oxidation in isolated working rat hearts reperfused after a period
	of transient global ischemia. Circ. Res. 66, 546-553
	46. Kantor, P.F., Lucien, A., Kozak, R., Lopaschuk, P.D. (2000) The antianginal
	drug trimetazidine shifts cardiac energy metabolism from fatty acid oxidation to
	glucose oxidation by inhibiting mitochondrial long chain 3-ketoacyl coenzyme
	A thiolase. Circ. Res. 86, 580-588
	47. Tynerir, B., Colak, O., Alatas, O., Besogul, Y., Kural, T., Aslan, R. (1999)
	Messurement of troponin T to detect cardioprotective effect of trimetazidine
	during coronary artery bypass-grafting. Ann. Thorac. Surg. 68, 2173-2176

Table 2 . Patient groups characteristic Treatment group

 2

	Variable	Placebo (n=121)	(n=117) Etomoxir 40mg	(n=108 ) Etomoxir 80mg	Overall
	Age (yr) (mean ±sd)	60.1±11.1	60.6±10.1	61.2±10.2	60.6±10.5
	Gender (male/female)	101/20	92/26	88/20	281/66
	% current smokers	15%	17%	11%	14%
	% Caucasian	98%	96%	98%	97%
	Systolic bp (mmHg) (mean±sd)	124.1±17.6	127.7±17.5	126.2±17.4	126.0±17.5
	Diastolic bp (mmHg) (mean±sd)	79.4±10.5	77.9±8.7	78.7±9.3	78.7±9.6
	Heart rate (bpm) (mean±sd)	74.3±13.7	74.0±12.8	76.1±15.0	74.8±13.8
	Weight (kg) (mean±sd)	80.9±11.1	79.6±12.3	82.1±17.4	80.9±13.8
	Height (cm) (mean±sd)	170.9±7.5	169.9±7.6	168.6±14.8	169.8±10.4
	LVEF (%)	30.5±5.9 (121)	31.0±6.3 (117) 30.5±6.3 (107)	30.7±6.1 (345)
	LVEDD (cm)	10.1±13.9 (121)	9.3±12.8 (116) 11.0±16.0 (104)	10.1±14.2 (341)
	Fractional shortening (%)	15.2±4.4 (61)	16.9±7.2 (68)	16.4±5.9 (53) 16.2±6.0 (182)
	Left ventricular wall thickness at the septum (mm)	10.6±2.3 (112)	11.3±2.5 (107) 11.5±2.7 (97) 11.1±2.5 (316)
	Left ventricular wall thickness at the posterior wall (mm)	10.6±2.1 (111)	10.5±2.1 (104) 10.9±1.9 (95)	10.7±2.1 (310)
	NYHA II	70	75	64	209
	NYHA III	51	42	44	137

Table 3 :

 3 Change from baseline in total exercise test time at week 24 endpoint (sec) Estimated from ANCOVA model with factors for treatment and country with baseline value as a covariate. A negative difference indicates the latter treatment is favoured

		Placebo		Etomoxir 40mg	Etomoxir 80mg
		(n=83)		(n=74)		(n=77)
	Baseline (mean±sd)	388.8±134.2	435.2±134.6	414.0±154.3
	Week 24 endpoint (mean±sd)	392.1±149.3	445.4±150.6	433.3±152.8
	Change from baseline (mean±sd)	3.3±92.2		10.2±73.5	19.4±61.8
	Pairwise comparisons	Mean difference	a	Se	95% CI	P
	Placebo-etomoxir 40mg	-10.9		12.4	-35.3,13.5	0.38	n = 16
	Placebo-etomoxir 80mg	-18.6		12.2	-42.5,5.4	0.13	n = 14
	Etomoxir 40mg-Etomoxir 80mg	-7.7		12.5	-32.3,17.0	0.54	n = 14
	A

Table 4 A: Change from baseline in six minutes walk test (m)

 4

		Placebo	Etomoxir 40 mg	Etomoxir 80 mg
		(n = 83)	(n = 74)	(n = 77)
	Baseline	401.2	382.2	392.5
	Change from	30.4 ± 95.9	22.7 ± 81.8	30.6 ± 52.7
	baseline (mean ±	(n = 19)	(n = 16)	(n = 12)
	sd)
		N.S.	N.S.	N.S.

Table 4 B: Change from baseline in left ventricular ejection fraction (%)

 4

		Placebo	Etomoxir 40 mg	Etomoxir 80 mg
	Baseline	30.5 ± 5.9	31.0 ± 6.3	30.5 ± 6.3
		(n = 121)	(n = 117)	(n = 104)
	Change from	5.8 ± 10.6	6.3 ± 11.1	8.2 ± 11.6
	baseline(mean±sd)	(n = 21)	(n = 17)	(n = 13)
		N.S.	N.S.	N.S.

Table 5 . Serious adverse events leading to withdrawal during the randomised phase (*accoring to the judgement of the local investigator)

 5

	Patient number	Adverse event	Severity *	Relationship to study medication
	Placebo
	12503	ventricular tachycardia	Moderate	Possible
	Etomoxir
	40mg
	12505	cardiac decompensation	Severe	Possible
	13003	highly elevated liver enzymes	Moderate	Probable
	13103	ventricular tachycardia	Severe	Unlikely
	14107	neoplasma of testes	Severe	Not related
	Etomoxir
	80mg
	10007	increase of transaminases,	Moderate	Possible
		elevated liver enzymes
	11105	elevation of liver enzymes	Unknown	Possible
	12504	arrhythmia	Moderate	Probable
	14105	elevation of liver enzymes	Moderate	Probable
	14106	angina pectoris	Moderate	Unlikely

Clinical Science Immediate Publication. Published on 23 Feb 2007 as manuscript CS20060307 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Table 6 : Four patients with elevated liver enzymes:

 6

Clinical Science Immediate Publication. Published on 23 Feb 2007 as manuscript CS20060307 © 2007 The Authors Journal compilation © 2007 Biochemical Society

© 2007 The Authors Journal compilation © 2007 Biochemical Society