Dr Prasad Gunaruwan
email: gunaruwan@doctors.org.uk

Abdul Maher

Lynne Williams

James Sharman

Matthias Schmitt

Ross Campbell

Michael Frenneaux

Effects of Bradykinin on Venous Capacitance in Health and Treated Chronic Heart Failure

Keywords: bradykinin, venous capacitance, heart failure, bradykinin receptors, ACE inhibitors, angiotensin receptor blockers

We investigated the effects of basal and intra-arterial infusion of bradykinin on unstressed forearm vascular volume (a measure of venous tone) and blood flow in health (G1, n=20) and in chronic heart failure patients treated with angiotensin converting enzyme (ACE) inhibitors (G2, n=16) and angiotensin receptor blockers (ARB) (G3, n=14). We used radionuclide plethysmography to examine the effects of bradykinin and of the bradykinin antagonists B9340 (B1/B2 antagonist) and HOE140 (B2 antagonist). Bradykinin infusion increased unstressed forearm vascular volume in a similar dose dependent manner in G1 and G3 (G1 maximum 12.3±2.1%; p<0.001 vs baseline, G3 maximum 9.3±3.3%; p<0.05 vs baseline, p=NS for difference) but the increase in unstressed volume in G2 was higher (maximum 28.8±7.8%; p<0.001 vs baseline; p<0.05 for the difference between groups). In contrast, while the increase in blood flow in G1 (maximum 362±9%; p<0.001) and in G2 (maximum 376±12%; p<0.001) was similar (p=NS for the difference between groups), the increase in G3 was less (maximum 335±7%; p<0.001; p <0.05 for the difference between groups). Infusion of each receptor antagonist alone similarly reduced basal unstressed volume and blood flow in G2 but not in G1 or G3. In conclusion, bradykinin does not contribute to basal venous tone in health, but in ACE inhibitor-treated chronic heart failure it does. In ARB-treated heart failure venous responses to bradykinin are preserved but arterial responses reduced compared with healthy controls.

Bradykinin mediated vascular responses in both health and heart failure are mediated by the B2 rather than the B1 receptor.

Introduction

Bradykinin is a potent endothelium-dependent dilator of the resistance vessels in health [1;2] and in chronic heart failure (CHF) [START_REF] Davie | Role of bradykinin in the vasodilator effects of losartan and enalapril in patients with heart failure[END_REF]. In patients treated with angiotensin converting enzyme (ACE) inhibitors, bradykinin contributes to the regulation of basal vascular tone in the resistance vessels [START_REF] Witherow | Bradykinin contributes to the vasodilator effects of chronic angiotensin-converting enzyme inhibition in patients with heart failure[END_REF] and in the pulmonary circulation [START_REF] Cruden | Bradykinin contributes to the systemic hemodynamic effects of chronic angiotensin-converting enzyme inhibition in patients with heart failure[END_REF]. In health bradykinin is also a potent dilator of conduit veins [START_REF] Dachman | Mechanism of bradykinin-induced venodilation in humans[END_REF]. However the physiology of small veins and venules that make up most of the venous volume differs markedly from that of conduit veins [START_REF] Schmitt | Assessment of venous capacitance. Radionuclide plethysmography: methodology and research applications[END_REF]. Since a large proportion of total blood volume is contained within these vessels even small changes in the tone of these 'capacitance' veins will influence cardiac pre-load. This is particularly relevant in CHF.

Understanding the receptors involved in mediating these effects is also important since they are potential therapeutic targets. To the best of our knowledge the direct local effects of bradykinin on capacitance veins have not been assessed in health or in chronic heart failure in man.

In health, bradykinin mediates its effects primarily via its constitutively expressed type 2 receptor (B2) [START_REF] Farmer | Biochemical and molecular pharmacology of kinin receptors[END_REF]. In arteries there is evidence to suggest that there is a proportionately higher expression of the bradykinin receptor type 1 (B1) in CHF [3;4], either due to up regulation of the B1 receptor or to down regulation of the B2 receptor [START_REF] Kuoppala | Down-regulation of cardioprotective bradykinin type-2 receptors in the left ventricle of patients with endstage heart failure[END_REF]. The contribution of B1 and B2 receptors to bradykinin-induced changes in venous capacitance has not been evaluated in health or in CHF. Furthermore, endothelial dysfunction affects arterial vessels in CHF and an impaired dilator response to bradykinin might be expected. In contrast, we have previously shown preservation of endothelium dependent responses to carbachol [START_REF] Nightingale | Preservation of venous endothelial function in the forearm venous capacitance bed of patients with chronic heart failure despite arterial endothelial dysfunction[END_REF] and also preservation of the nitric oxide-dependent response to atrial natriuretic peptide [START_REF] Schmitt | Atrial natriuretic peptide regulates regional vascular volume and venous tone in humans[END_REF], in the forearm capacitance bed of patients with CHF despite attenuation of responses in arteries. Preservation of responses to bradykinin in capacitance vessels might thus be anticipated in CHF.

To assess the role of bradykinin and to identify the receptors involved in mediation of its effects on capacitance veins in both health and in treated CHF, we tested three hypotheses. First, that bradykinin modulates basal forearm venous capacitance in CHF patients treated with ACE inhibitors but not in health or in CHF patients treated with angiotensin receptor blockers (ARB) .

Secondly, we hypothesised that in CHF patients the response to bradykinin is impaired in resistance vessels but preserved in capacitance vessels. Thirdly, that B1-mediated effects are greater in both resistance and capacitance vessels in CHF patients when compared to healthy controls. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Subjects and Methods

We studied 20 healthy volunteers and 30 patients with stable treated CHF associated with left ventricular systolic dysfunction (ejection fraction <40%) who had NYHA class II-III symptoms.

16 patients were on maximal tolerated doses of ACE inhibitors and 14 patients were on maximally tolerated doses of ARB. Medications and doses are listed in Table1. All subjects were on an ad-libitum diet, but refrained from caffeine for at least 12 hours prior to the study.

Smoking was an exclusion criterion for the volunteers, and there were no current smokers in the patient group. All medications were withheld for at least 24 hours. Patient and subject characteristics are summarised in Table 1. Written informed consent was provided by all subjects, and the study was approved by the local research ethics committee.

Subjects rested supine in a temperature controlled laboratory (21-22°C). An 18-gauge cannula was inserted into a vein in the ante cubital region of the dominant arm and radio-labelling of red blood cells was carried out as described previously [START_REF] Schmitt | Assessment of venous capacitance. Radionuclide plethysmography: methodology and research applications[END_REF]. A 27-gauge arterial needle (Coopers Engineering, Birmingham) mounted onto a 16-gauge epidural catheter was then inserted into the non-dominant brachial artery, and kept patent by continuous infusion of 0.9% saline. Following 20 minutes rest and a 15-minute intra-brachial 0.9% saline infusion, baseline forearm blood flow (FBF) and forearm venous volume (FVV) was measured. FBF was measured using standard mercury in silastic strain gauge plethysmography (Hokanson, Bellevue, WA, USA), as described elsewhere [12;13]. The changes in the infused arm were corrected for changes in the control arm.

FVV was measured by combining venous occlusion plethysmography, and equilibrium bloodpool scintigraphy, as described previously [7;11]. In brief: following modified ex-vivo radiolabelling of red cells with Technetium (Tc m99 ), blood pool volume/pressure relations were constructed for both forearms, by inflating upper arm cuffs to 10, 20 and 30mmHg for one minute at each venous occlusion pressure. Dynamic images were acquired continuously, firstly during infusion of normal saline and then during each of the infusions as described in the experiments below. After correction for physical decay, scintigraphic vascular volume was plotted against occluding cuff pressure. Linear regression was performed and a linear model was adopted if the R 2 value was >0.9. Parallel shifts of the relation indicate a change in venous tone.

The intercept of the regression curve with the Y-axis reflects 'unstressed' venous volume (i.e. the volume that would exist at a theoretical zero transmural pressure gradient). The unstressed volume during normal saline infusion was arbitrarily denoted as 100%, and subsequent readings A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

were expressed as a percentage of this value. Changes in the infused arm were then corrected for those occurring in the control arm.

Measurements of FBF and FVV were repeated after each infusion of bradykinin and receptor antagonists in the experiments described below.

Vascular response to exogenous bradykinin: In all subjects, bradykinin (Clinalfa, Switzerland) was then infused incrementally at 31.8 ng/min (30 pmol/min) and 318 ng/min (300 pmol/min) for 6 minutes at each dose.

Assessment of antagonism of exogenous bradykinin:

We studied the effects of the specific B2 receptor antagonist HOE140 (Clinalfa, Switzerland) in 12 healthy volunteers and 15 patients (n=10 on ACE inhibitors and n=5 on ARB) and the non-specific B1/B2 receptor antagonist B9340 (Clinalfa, Switzerland) in 8 healthy volunteers and 15 patients (n=6 on ACE inhibitors and n=9 on ARB). Following assessment of vascular response to exogenous bradykinin, HOE140 or B9340 was co-infused at 13.5 nmol/min (17.6 μg/min and 17.8 μg/min respectively) with bradykinin 318ng/min (300 pmol/min) for 6 minutes. A 40:1 antagonist to bradykinin ratio was maintained to ensure adequate local antagonist concentration.

Assessment of endogenous bradykinin activity:

Following assessment of the effects of the receptor antagonists, 0.9% saline was infused, and measurements were allowed to return to baseline and equalise between the infused and control arms. HOE140 or B9340 was then infused at 13.5 nmol/min (17.6 μg/min and 17.8 μg/min respectively) for another 6 minutes.

Blood pressure was monitored continuously with finger photo-plethysmography (TNO-TPD Biomedical Instrumentation, Amsterdam, Netherlands). Heart rate was recorded throughout the study via a 3 lead ECG. (p) value of <0.05 was considered significant. Within each subject group (controls, ARB-treated CHF, ACE inhibitor-treated CHF), one-way analysis of variance was carried out for the absolute FBF ratios between the infused and the control arms for the analysis of FBF response to bradykinin. Two-way analysis of variance was performed to assess between group differences and Bonferroni correction was applied for multiple comparisons. One-way analysis of variance was carried out for the % changes of unstressed FVV between the infused arm and the control arm for the analysis of unstressed FVV response to bradykinin, and two-way analysis of variance was performed to assess between group differences. Two-way analysis of covariance (ANCOVA) was carried out for the analysis of the two antagonists B9340 and HOE140, between each pair of the three subject groups, using the FBF and unstressed FVV differences at maximum bradykinin induced dilatation as covariate. A paired sample t-test was used for the analysis of basal bradykinin effects within each group.

Results

Subject characteristics are shown in Table 1.

Blood pressure and heart rate did not change significantly from baseline during or at the end of the infusions (Baseline blood pressure±SEM 120/65±4/4, 110/60±8/6 and 115/64±12/8mmHg for volunteers, ACE inhibitor-treated CHF and ARB-treated CHF respectively vs blood pressure during final infusion±SEM 118/65±6/4, 118/58±14/10 and 112/60±16/12 mmHg respectively for the groups above).

Effects of bradykinin infusion on resistance vessels:

FBF increased significantly in the infused vs non-infused arms in healthy subjects and in both CHF groups (ACE inhibitor-treated and ARB-treated) (see Table 2). The increase in FBF in healthy volunteers and ACE inhibitortreated CHF patients was similar, but both were significantly higher (p<0.05, two-way ANOVA) than in ARB-treated CHF patients (Figure 1).

Effects of bradykinin infusion on capacitance vessels:

Unstressed FVV increased significantly in all 3 groups. The percentage increases from baseline are summarised in Table 2.

The unstressed FVV increase in ACE inhibitor-treated CHF patients was significantly higher (p<0.05, two-way ANOVA) than in both healthy volunteers and ARB-treated CHF patients (Figure 2).

Co-infusion of bradykinin with receptor antagonists: B9340 and HOE140 both attenuated the

FBF and unstressed FVV responses to 318ng/min (300 pmol/min) infusion of bradykinin, to a similar extent in all 3 groups (P<0.05, ANCOVA; Figures 3 and4). There was no significant difference between B9340 and HOE140 (P>0.05 ANCOVA).

Baseline bradykinin activity:

Discussion

The primary focus of bradykinin related research in the past has been on the peripheral resistance vasculature [START_REF] Cockcroft | Comparison of angiotensin-converting enzyme inhibition with angiotensin II receptor antagonism in the human forearm[END_REF][START_REF] Cockcroft | Inhibition of bradykinin-induced vasodilation in human forearm vasculature by icatibant, a potent B2-receptor antagonist[END_REF][START_REF] Davie | Role of bradykinin in the vasodilator effects of losartan and enalapril in patients with heart failure[END_REF][START_REF] Witherow | Bradykinin contributes to the vasodilator effects of chronic angiotensin-converting enzyme inhibition in patients with heart failure[END_REF], the coronary arteries [START_REF] Groves | Role of endogenous bradykinin in human coronary vasomotor control[END_REF], and the pulmonary circulation [START_REF] Cruden | Bradykinin contributes to the systemic hemodynamic effects of chronic angiotensin-converting enzyme inhibition in patients with heart failure[END_REF]. A number of studies have examined the effects of bradykinin on the dorsal hand vein [6;15]. However it is increasingly clear that such conduit veins may have different physiological characteristics to the small veins and venules that contribute most to the capacitance vasculature [START_REF] Schmitt | Assessment of venous capacitance. Radionuclide plethysmography: methodology and research applications[END_REF]. Although Mason et al [START_REF] Mason | Effects of bradykinin on forearm venous tone and vascular resistance in man[END_REF] examined the effects of systemic infusions of bradykinin on venous capacitance, two important caveats should be considered. First, systemic infusions of bradykinin lead to stimulation of baro-reflexes and other peripheral and systemic compensatory responses. Indeed, there is evidence that bradykinin may alter baro-reflex sensitivity [START_REF] Bomtempo | Interaction of bradykinin and angiotensin-(1-7) in the central modulation of the baroreflex control of the heart rate[END_REF].

Second, venous capacitance was measured using strain gauge venous occlusion plethysmography. Bradykinin is known to affect capillary permeability, thus interpretation of limb volume changes as being due to changes in vascular volume may be very misleading [START_REF] Schmitt | Assessment of venous capacitance. Radionuclide plethysmography: methodology and research applications[END_REF].

To our knowledge, this present study is the first to directly measure changes in venous tone and regional vascular volume in response to local infusions of bradykinin in healthy subjects. In addition to demonstrating modulation of FVV by exogenous bradykinin, our results demonstrate that endogenous bradykinin does not contribute to the regulation of basal resistance or capacitance vascular tone in the human forearm in health. We also confirm that in health, bradykinin exerts its effects on both resistance and capacitance vessels through the B2 receptor.

In CHF patients treated with ACE inhibitors forearm resistance vessel responses to bradykinin were similar to those of healthy controls, but in patients treated with ARB they were significantly reduced. The most likely explanation for these observations is that endothelial dysfunction in CHF patients reduces responsiveness of resistance vessels to bradykinin, and that ACE inhibitor treatment increases local concentrations to a greater extent than ARB (by inhibition of bradykinin breakdown), resulting in 'preservation' of the response to infused bradykinin. ACE inhibitors and ARB may ameliorate endothelial dysfunction in some disease states [18;19], but they do not normalise it. Another possible explanation is that B2 receptors are 're-sensitized' in the presence of ACE inhibitors via the influences of angiotensin 1-9 and angiotensin 1-5 [START_REF] Chen | Hydrolysis of angiotensin peptides by human angiotensin I-converting enzyme and the resensitization of B2 kinin receptors[END_REF]. Our finding of preservation of resistance vessel response to exogenous bradykinin in ACE inhibitor treated patients is consistent with those of Maguire et al [START_REF] Maguire | Bradykinin infusion in chronic cardiac failure and the effects of captopril[END_REF], but our finding that responses in ARB-treated patients are impaired is, to the best of our knowledge novel. Davie et al [START_REF] Davie | Role of bradykinin in the vasodilator effects of losartan and enalapril in patients with heart failure[END_REF] observed reduced responsiveness in CHF patients treated with ARB vs those treated with ACE inhibitors but there was no healthy control group comparator. Importantly, in contrast with our findings in resistance vessels, we found that venous responses to bradykinin in CHF patients treated with ARB were similar to those of healthy controls, and that responses in the ACE inhibitor treated patients were higher than those of healthy controls.

Although the mechanism is unproven we propose that this observation is most likely due to preservation of endothelial function in capacitance vessels in CHF patients. We previously demonstrated that in ACE inhibitor-treated CHF patients, there was marked arterial endothelial dysfunction, but preservation of endothelium dependent responses to carbachol in the capacitance vessels [START_REF] Nightingale | Preservation of venous endothelial function in the forearm venous capacitance bed of patients with chronic heart failure despite arterial endothelial dysfunction[END_REF]. Furthermore in another study we showed that much of the responses of forearm resistance and capacitance vessel response to atrial natriuretic peptide is mediated via endothelium dependent nitric oxide (NO) release and that in CHF there was blunting of this NO dependent component in the resistance, but not the capacitance vessels [START_REF] Schmitt | Effects of exogenous and endogenous natriuretic peptides on forearm vascular function in chronic heart failure[END_REF].

There is histological evidence that the expression of B1 and B2 receptors may shift towards preferential B1 expression in CHF, with a reduction in B2 receptor expression in end stage CHF [START_REF] Kuoppala | Down-regulation of cardioprotective bradykinin type-2 receptors in the left ventricle of patients with endstage heart failure[END_REF]. The studies of Davie et al [START_REF] Davie | Role of bradykinin in the vasodilator effects of losartan and enalapril in patients with heart failure[END_REF] and Witherow et al [START_REF] Witherow | Bradykinin contributes to the vasodilator effects of chronic angiotensin-converting enzyme inhibition in patients with heart failure[END_REF] together indicate that bradykinin exerts basal effects on forearm resistance vessels in CHF patients treated with ACE inhibitors and that these effects are mediated via B1 rather than B2 receptors. In contrast, in a study using B1 receptor agonists, Cruden et al [START_REF] Cruden | B1 kinin receptor does not contribute to vascular tone or tissue plasminogen activator release in the peripheral circulation of patients with heart failure[END_REF] refuted any significant resistance vessel role for the B1 receptor in CHF. Our findings are consistent with the latter and demonstrate that both receptor antagonists (B9340 and HOE140) negated the effects of exogenous bradykinin equally, in resistance and capacitance vessels of the forearm to a similar extent. Most of our patients were only moderately symptomatic (NYHAII). Most of Cruden et al [START_REF] Cruden | B1 kinin receptor does not contribute to vascular tone or tissue plasminogen activator release in the peripheral circulation of patients with heart failure[END_REF] patients also had relatively mild (NYHA II) symptoms. In contrast, patients in Witherow at al [START_REF] Witherow | Bradykinin contributes to the vasodilator effects of chronic angiotensin-converting enzyme inhibition in patients with heart failure[END_REF] study were mainly severely symptomatic (NYHA III or IV). Thus our patient group may not manifest the shift of receptor expression. We draw the conclusion that in treated CHF patients with moderate symptomatic limitation the effects of exogenous bradykinin are primarily mediated via the B2 receptor. It is possible that in more severe heart failure B1 mediated effects may be more important.

We also show that endogenous bradykinin contribute to basal venous tone in ACE inhibitortreated CHF as well as confirming the previous findings [START_REF] Witherow | Bradykinin contributes to the vasodilator effects of chronic angiotensin-converting enzyme inhibition in patients with heart failure[END_REF] that endogenous bradykinin contribute to modulation of the basal forearm resistance in ACE inhibitor-treated CHF.

However, in contrast to Witherow et al [START_REF] Witherow | Bradykinin contributes to the vasodilator effects of chronic angiotensin-converting enzyme inhibition in patients with heart failure[END_REF], our results show that these basal effects were also antagonised by HOE140 and B9340 to a similar extent, reinforcing our finding that B2 receptor remains to be the expressed receptor type in moderate treated CHF. It has been suggested that due to inhibition of prostaglandin synthesis, aspirin might attenuate the beneficial effects of ACE inhibitors [START_REF] Cleland | Does aspirin attenuate the effect of angiotensin-converting enzyme inhibitors in hypertension or heart failure?[END_REF]. Several clinical trials have shown that aspirin treatment may increase hospital admissions due to worsening heart failure [24;25;25;26]. However, the balance of available evidence suggests that low dose aspirin does not impair the arterial dilatation induced by ACE inhibitors in CHF [START_REF] Davie | Effect of aspirin on vasodilation to bradykinin and substance P in patients with heart failure treated with ACE inhibitor[END_REF], nor does it blunt the beneficial effects on mortality [28;29]. Low dose aspirin inhibits thromboxane formation and has little or no effect on prostacyclin PGI 2 , the cyclo-oxygenase dependent second messenger for bradykinin [START_REF] Griffoni | Selective inhibition of prostacyclin synthase activity by rofecoxib[END_REF]. In addition, any effect on PGI2 is short lasting (less then 6 hours) [31;32] in comparison to effects on thromboxane. We did not exclude patients who had been taking low dose aspirin (<150mg) because the ethics committee considered it unethical to do so, but we ensured that aspirin was withheld for at least 48 hours prior to the experiments, by which time vascular effects would have ceased.

The objective of the present study was to show that bradykinin is an important mediator of venous tone in health and in CHF. However, in dissecting the mechanisms involved in mediating the effects that we have shown, our studies are limited by the lack of direct assessment of the nitric oxide dependent component. However, addition of another infusion step with N Gmonomethyl-L-arginine (LNMMA) would have extended the studies beyond 3 hours in duration. We have found it impossible for subjects to remain still and relaxed beyond 3 hours with an arterial needle in-situ. No dose comparison studies exist between ACE inhibitors and ARB. Thus we cannot quantitatively determine if the doses that our patients were on inhibited the angiotensin-aldosterone axis to a comparable degree. This weakness needs to be considered when comparing the differences we identified between ACE inhibitors and ARB. Furthermore, as the patients were already on either ACE inhibitors or ARB, it is possible, at least in theory, that baseline differences in physiology may have existed prior to initiation of treatment.

In conclusion, we demonstrate that the FBF response to bradykinin is impaired in ARB-treated CHF, but is not in ACE inhibitor-treated patients. We also demonstrate that forearm venous capacitance is increased by exogenous bradykinin in both health and in CHF and that the response is preserved in ARB-treated patients and increased in ACE inhibitor-treated patients with CHF vs healthy controls. Resistance and capacitance vessel effects are mediated via the B2 receptor in both health and in CHF. Bradykinin does not play a role in modulating basal forearm venous tone in health, but contributes to the basal forearm venous tone in ACE inhibitor-treated and not in ARB-treated patients with CHF. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Clinical

  Science Immediate Publication. Published on 18 Sep 2008 as manuscript CS20080096 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080096

Clinical

  Science Immediate Publication. Published on 18 Sep 2008 as manuscript CS20080096 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080096 A c c e p t e d M a n u s c r i p t Statistics Sample size was based on sample size calculations with 80% power to detect a 10% change in unstressed FVV with significance at 0.05 level with a sigma of 10%. Statistical analysis was performed using SPSS version 11.5.1. All data are expressed as mean±SEM and a probability

  Clinical Science Immediate Publication. Published on 18 Sep 2008 as manuscript CS20080096 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080096A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  Infusion of B9340 or HOE140 did not reduce the FBF or unstressed FVV in healthy volunteers or in ARB-treated patients (p>0.05, Paired t-test; Figures 5, 6, 7 and 8). For HOE140 the percentage changes in FBF±SEM were -4.4±11.2% and 4.6±12.8% and percentage changes in unstressed FVV±SEM were -0.4±1.8% and -0.7±1.9% respectively (p>0.05, Paired t-test) for normal volunteers and for ARB-treated patients. However, both B9340 and HOE140 reduced FBF and unstressed FVV in ACE inhibitor-treated patients (p<0.05, paired t-test; Figures 5, 6, 7 and 8). For HOE140 the percentage change in FBF±SEM was -27.8±10.8% (p>0.05, Paired t-test) and percentage change in unstressed FVV±SEM was -4.0±1.8% (p>0.05, Paired t-test) ACE inhibitor-treated patients. Clinical Science Immediate Publication. Published on 18 Sep 2008 as manuscript CS20080096 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080096 A c c e p t e d M a n u s c r i p t

Clinical

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Clinical

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Clinical

  Science Immediate Publication. Published on 18 Sep 2008 as manuscript CS20080096 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080096 A c c e p t e d M a n u s c r i p t

Figure 1 Figure 2 ClinicalFigure 3 .

 123 Figure 1

Figure 4 .

 4 Figure 4.

Figure 5 Figure 6 Clinical

 56 Figure 5

Figure 7 Figure 8 Clinical

 78 Figure 7

Figure Legend Figure 1 .

 Legend1 Figure Legend

Figure 2 .

 2 Figure 2. Changes in FVV, percentage of baseline in response to bradykinin. BK 30 = FBF during the infusion of Bradykinin at 30 pmol/min and BK 300 = FBF during the infusion of bradykinin at 300 pmol/min. ACEI = ACE inhibitor-treated CHF patients and ARB = CHF patients treated with ARB. * Denotes significant difference, p<0.05 (two-way ANOVA).

Figure 3 .

 3 Figure 3. Percentage changes in the FBF ratio between the infused and control arms during infusion of bradykinin alone and co-infusion with B9340 or HOE140. BK 300 = FBF during the infusion of bradykinin at 300 pmol/min. ACEI = ACE inhibitor-treated CHF patients and ARB = CHF patients treated with ARB. * Denotes significant difference, p<0.05 (two-way ANCOVA).

Figure 4 .

 4 Figure 4. Changes in FVV, percentage of baseline during infusion of bradykinin alone or coinfusion with B9340 or HOE140. BK 300 = FVV during the infusion of bradykinin at 300 pmol/min. ACEI = ACE inhibitor-treated CHF patients and ARB = CHF patients treated with ARB. * Denotes significant difference, p<0.05 (two-way ANCOVA).

Figure 5 .

 5 Figure 5. Percentage changes in the FBF ratio between the infused and control arms during infusion of B9340, after the period of normal saline washout. ACEI = ACE inhibitor-treated CHF patients and ARB = CHF patients treated with ARB. * Denotes significant difference, p<0.05 (paired t-test).

Figure 6 .

 6 Figure 6. Changes in FVV, percentage of baseline during infusion of B9340 after the period of normal saline washout. ACEI = ACE inhibitor-treated CHF patients and ARB = CHF patients treated with ARB. * Denotes significant difference, p<0.05 (paired t-test).

Figure 7 .

 7 Figure 7. Percentage changes in FBF during infusion of B9340 or HOE140 in ACE inhibitortreated CHF patients after the period of normal saline washout. * Denotes significant difference, p<0.05 (paired t-test).

Figure 8 .

 8 Figure 8. Changes in FVV, percentage of baseline during infusion of B9340 or HOE140 in ACE inhibitor-treated CHF patients after the period of normal saline washout. * Denotes significant difference, p<0.05 (paired t-test).

  Clinical Science Immediate Publication. Published on 18 Sep 2008 as manuscript CS20080096 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080096

Table 1 . Subject Characteristics

 1

	Clinical Science Immediate Publication. Published on 18 Sep 2008 as manuscript CS20080096
	Parameter	Volunteers	CHF+ACEI	CHF+ARB
	Number	20	16	14
	Age (range) years	58 (52-68)	64 (50-80)	66 (50-80)
	Sex, male:female Etiology, IHD:DCM NYHA class II:III Treated hypertension Treated diabetes Medications Beta blockers Diuretics Aspirin Nitrate Lipid lowering Digoxin Warfarin Ramipril 10mg Ramipril 5mg Perindopril 4mg Lisinopril 20mg Lisinopril 2.5mg Fosinopril 40mg Losartan 50mg Candesartan 8mg Losartan 100mg Losartan 25mg Valsartan 160mg MUGA EF % Baseline FBF ml/min Baseline FVV (counts) Values are numbers (Male:Female or IHD:DCM) or range (age range) or Mean±SEM. 11:9 14:2 8:6 11:5 12:2 14:2 11:3 8 6 4 3 10 11 12 12 11 3 5 6 11 10 3 3 6 5 5 4 3 2 1 1 4 4 2 2 2 54±1 38±2 35±3 1.8±0.2 1.7±0.2 1.7±0.3 956±50 899±148 947±41 CHF+ACEI = ACE inhibitor-treated CHF patients; CHF+ARB = ARB-treated CHF patients. Total daily doses of the drugs are listed. MUGA,EF% indicates left ventricular ejection fraction measured by multiple gated scanning. Baseline FBF is for the infused arm in mls/min/100ml of A c c e p t e d M a n u s c r i p t	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080096
	forearm volume. Baseline FVV is in radioactive counts in the infused forearm.

Table 2 . Changes in FBF and FVV with bradykinin (corrected for control arm) FBF FVV

 2

	Clinical Science Immediate Publication. Published on 18 Sep 2008 as manuscript CS20080096
	Infusion	Volunteer CHF+ACEI	CHF+ARB	Volunteer CHF+ACEI CHF+ARB
	Bradykinin 31.8 ng/min	231±9 ‡	262±11 ‡	220±10 ‡	2.7±1.6	11.2±3.1*	1.1±2.1
	Bradykinin 318 ng/min	362±9 †	376±12 †	335±17 †	12.3±2.1 †	28.8±7.8 †	9.3±3.3*
	Values are Mean±SEM indicating percentage changes from baseline. CHF+ACEI = ACE
	Inhibitor-treated CHF patients; CHF+ARB = ARB-treated CHF patients. Significant changes from baseline are indicated by * p<0.05, ‡ p<0.01, † p<0.001 (One-way ANOVA).

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

© 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgements

This work was supported by the British Heart Foundation and by M.G. Cezar memorial scholarships from the University of Tasmania, Australia. Clinical Science Immediate Publication. Published on 18 Sep 2008 as manuscript CS20080096 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080096

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

This work was supported by the British Heart Foundation


Angela Beatrice Scardovi
email: ab.scardovi@libero.it

Renata De Maria

Andrea Celestini

Silvia Perna

Claudio Coletta

Mauro Feola

Nadia Aspromonte

Gianluca Rosso

Alessandro Carunchio

Alessandro Ferraironi

Silvia Perna°

Gian Luca Rosso

Alessandro Pimpinella

Roberto Ricci

Dott Angela

Beatrice Scardovi

The additive prognostic value of the cardiopulmonary exercise test in elderly patients with heart failure

Keywords: Elderly, Heart Failure, Cardiopulmonary exercise test, Preserved left ventricular ejection fraction heart failure

Introduction

Heart failure (HF) is a common chronic diseases in the elderly. While relief of symptoms and acceptable quality of life are the main goals of treatment in frail patients with multiple disabling comorbidities, "robust" elderly subjects, with no relevant strength or mobility problems, may have an outlook similar to younger patients, and should be carefully assessed for up-to-date treatment.

When assessing prognosis in elderly HF patients, certain predictors are particularly relevant, in view of their prevalence in older age. Renal dysfunction, common in the elderly and frequent in HF, increases mortality across the whole spectrum of ventricular impairment [START_REF] Mcalister | Renal insufficiency and heart failure: prognostic and therapeutic implications from a prospective cohort study[END_REF][START_REF] Hillege | Renal function as a predictor of outcome in a broad spectrum of patients with heart failure[END_REF]. In the elderly, HF with preserved left ventricular ejection fraction (PLVEF-HF) is a common and costly clinical entity, with mortality and one-year readmission rates [START_REF] Hogg | Heart failure with preserved left ventricular systolic function ; epidemiology, clinical characteristics and prognosis[END_REF][START_REF] O'conner | Clinical characteristics and long -term outcomes of patients with heart failure and preserved systolic function[END_REF] similar to HF with depressed systolic function (SHF). Increasing evidence links diastolic dysfunction to exercise intolerance [START_REF] Packer | Abnormalities of diastolic function as a potential cause of exercise intolerance in chronic heart failure[END_REF] .

Among laboratory investigations, the cardiopulmonary exercise test with gas exchange measurement (CPET), when technically feasible, is an accurate tool in routine clinical evaluation and risk stratification of HF patients [START_REF]Working Group on Cardiac Rehabilitation and Exercise Physiology and Working Group on Heart Failure of European Society of Cardiology. Recommendations for exercise testing in chronic heart failure patients[END_REF][START_REF] Lang | Prognostic significance and measurement of exercise-derived hemodynamic variables in patients with heart failure[END_REF]. Reduced peak oxygen consumption (PVO2) and ventilatory abnormalities, such as an enhanced ventilatory response to exercise (EVR) provide objective and non-invasive evaluation of functional capacity and were shown to predict prognosis in a large subset of SHF and PLVEF-HF patients [START_REF] O'neill | Peak oxygen consumption as a predictor of death in patients with heart failure receiving beta-blockers[END_REF][START_REF] Corra | Ventilatory response to exercise improves risk stratification in patients with chronic heart failure and intermediate functional capacity[END_REF][START_REF] Arena | Peak VO2 and VE/VCO2 slope in patients with heart failure: a prognostic comparison[END_REF][START_REF] Guazzi | Cardiopulmonary exercise testing in the clinical and prognostic assessment of diastolic heart failure[END_REF]. Our group previously demonstrated the feasibility and safety of CPET in elderly HF patients [START_REF] Scardovi | Cardiopulmonary exercise test is safe and reliable in elderly patients with chronic heart failure[END_REF].

Brain natriuretic peptide (BNP), a composite marker of systolic and diastolic function and neurohormonal activation, is a prominent prognostic parameter, even in elderly patients [START_REF] Silver | Consensus panel 2004: a clinical approach for the diagnostic, prognostic, screening, treatment monitoring, and therapeutic roles of natriuretic peptides in cardiovascular diseases[END_REF][START_REF] Feola | Prognostic value of plasma brain natriuretic peptide, urea nitrogen, and creatinine in outpatients >70 years of age with heart failure[END_REF]. Recent clinical reports have focused on the prognostic usefulness of BNP and PVO 2 in HF patients. [START_REF] Isnard | Combination of B-type natriuretic peptide and peak oxygen consumption improves risk stratification in outpatients with chronic heart failure[END_REF][START_REF] De Groote | B-type natriuretic peptide and peak exercise oxygen consumption provide independent information for risk stratification in patients with stable congestive heart failure[END_REF], yet CPET-derived parameters of ventilatory efficiency may be superior to BNP in predicting prognosis in SHF patients [START_REF] Scardovi | Multiparametric risk stratification in patients with mild to moderate chronic heart failure[END_REF].

Aim of our study was to address the additive prognostic role of ventilatory parameters to clinical, neurohormonal and echocardiographic variables in stable elderly outpatients with mild to moderate SHF and PLVEF-HF on optimized medical treatment, followed-up at a dedicated HF clinic.

Methods

Study population

Between October 2001 and January 2006, 331 consecutive outpatients aged > 70 years with a diagnosis of HF since at least 3 months were screened for CPET evaluation and 95 were excluded for reasons detailed in Figure 1. Patients were in NYHA class I-III on stable drug doses without worsening symptoms or need for intravenous inotropic support during the last 4 weeks. Among the 236 consenting subjects who were able to perform a CPET test, 220 with a PVO2 < 25 mg/kg/min and a respiratory exchange ratio (RER) > 1.00 at CPET, indicating the achievement of anaerobic exercise conditions, were enrolled; 16 subjects with a RER ≤ 1 were excluded.

The study protocol was approved by the Institutional Ethics Committee and complies with the ethical rules for human experimentation that are stated in the Declaration of Helsinki.

Baseline laboratory assessment was performed according to standard laboratory methods.

Creatinine clearance was determined using the Cockcroft e Gault equation.

Patients were followed up at the outpatient HF clinic of our hospital; outcome data of patients who did not attend their scheduled appointments (<10% of those enrolled) were obtained by telephone interview of the patient or his/her family; none of these patients had A c c e p t e d M a n u s c r i p t been admitted to hospital. The primary combined study end-point was defined as the occurrence of death and hospital admission for worsening HF, arrhytmias or acute coronary syndromes .

Exercise test

A standard bicycle exercise ramp protocol was used, with increments of 10 watts/min. Details of test protocol have been published previously [START_REF] Scardovi | Cardiopulmonary exercise test is safe and reliable in elderly patients with chronic heart failure[END_REF][START_REF] Scardovi | Multiparametric risk stratification in patients with mild to moderate chronic heart failure[END_REF]. Before each test, oxygen and carbon dioxide analyzers and a flow mass sensor were calibrated by use of available precision gas mixtures and a 3-L syringe, respectively. To stabilize gas measurement, patients were asked to remain still for at least 3 minutes before beginning an upright graded bicycle exercise testing, using a continuous protocol, which was continued until exhaustion.

A 12-lead electrocardiogram was continuously recorded (MAX-1, Marquette Electronics, Milwaukee, Wis, USA). Blood pressure was recorded every minute by a cuff sphygmomanometer. Indications to stop the test were exhaustion, dyspnoea, angina, STsegment depression > 3 mm, high-degree AV block or ventricular tachycardia > 5 consecutive beats at a rate > 120/min, new-onset atrial fibrillation, or systolic blood pressure > 260 mmHg, as well as a progressive decrease in blood pressure.

Oxygen uptake (VO 2 ), carbon dioxide production (VCO 2 ) and minute ventilation were measured by breath-by-breath gas analysis with use of a computerized metabolic cart (V max 29, Sensormedics, Yorba Linda, Ca, USA). PVO 2 was recorded as the mean value of VO 2 during the last 30 seconds of the test and expressed as mL per kilogram per minute. PVO 2 was measured and expressed as absolute and percent predicted value from the individual age, sex, and body weight-corrected mean normal values (% PVO 2 ). The % PVO 2 was calculated for each patient using Wasserman's equation.

The O 2 uptake at the anaerobic threshold was determined by use the V-slope method [START_REF] Cohen-Solal | Multicentre study of the determination of peak oxygen uptake and ventilatory threshold during bicycle in chronic heart failure[END_REF].

Other considered parameters were: a) ventilation (VE) and expired O 2 ratio (VE/VO 2 ), b) VE/CO 2 production ratio (VE/VCO 2 ), (VE, VO 2 and VCO 2 L/min); c) VE/VCO 2 slope, calculated in every subject as the slope of the regression line relating VE to CO 2 output during exercise testing, with the exclusion of the final non-linear portion after the onset of acidotic drive to ventilation.

A VCO 2 /VO 2 ratio > 1.00 was selected for the definition of maximal exercise.

The VE/VCO 2 slope was used as the main index of the ventilatory response to exercise.

We defined EVR as a VE/VCO 2 slope ≥ 34 [START_REF] Ponikowski | Enhanced ventilatory response to exercise in patients with chronic heart failure and preserved exercise tolerance. Marker of abnormal cardiorespiratory reflex control and predictor of poor prognosis[END_REF]. The ratio of VE/VCO 2 slope to PVO 2 , previously suggested as a better predictor of prognosis than either variable alone [START_REF] Guazzi | Normalization for peak oxygen uptake increases the prognostic power of the ventilatory response to exercise in patients with chronic heart failure[END_REF] was also analysed.

Doppler echocardiography

Doppler echocardiography was performed using a Philiphs MS Sonos 5.500 D device equipped with a multiband 1.8-3.6 MHz probe. Left ventricular ejection fraction (LVEF) was measured by two blinded expert operators according to the modified Simpson's biplane method, in the apical-four and two-chamber views. Biplane volumes were calculated from area tracings using the disc summation method (modified Simpson's rule). Pulmonary systolic pressure (PAP) was calculated by means of right ventricular/ right atrial gradient in patients with detectable tricuspid regurgitation (peak ventricular/atrial gradient + 8-10 mmHg).

Transmitral flow velocity was determined by the pulsed-wave Doppler approach using a 2-4 mm sample volume placed at the mitral leaflet tips level in the apical four-chamber view. The transmitral pulsed-wave Doppler blood flow velocities were calculated off-line from 5 consecutive cardiac cycles to measure early diastolic (E) peak velocity (cm/sec), late diastolic (A) peak velocity (cm/sec), E/A velocity ratio and E velocity deceleration time (msec). A restrictive filling pattern was defined as presence of a E/A ratio > 2.0 cm/sec and a E deceleration time < 140 ms. In patients with atrial fibrillation, the restrictive filling pattern was defined by a E deceleration time < 140 ms calculated off-line from 10 consecutive cardiac cycles. Preserved left ventricular ejection fraction (PLVEF) was defined as LVEF ≥ 40% [START_REF] Permenkil | Course and prognosis in patients > 70 years of age with congestive heart failure and normal versus abnormal left ventricular ejection fraction[END_REF][START_REF] Smith | Outcomes in heart failure patients with preserved ejection fraction. Mortality, readmission and functional decline[END_REF][START_REF] Ahmed | Outcomes in ambulatory chronic systolic and diastolic heart failure: a propensity score analysis[END_REF].

Measurement of BNP plasma levels

Before exercise test, blood was collected by venipuncture from an antecubital vein, after 10 min of rest in the sitting position. A point-of-care test (Triage BNP test, Biosite Diagnostics®), based on immunofluorometric assay for quantitative determination of BNP in whole blood or plasma was used. Normality range values are 0-40 pg/mL. BNP concentrations were determined by nurses blinded to the cardiopulmonary exercise test results.

Statistical analysis

Categorical variables are presented as frequency percents and continuous variables as median values [interquartile range]. Between-group differences were tested by chi-square test and Student's t-test or non parametric testing according to normal or non normal variable distribution. A p value < 0.05 was considered statistically significant.

To determine the independent predictors of the primary combined end point of occurrence of death and hospital admission for major cardiovascular events, we entered variables significant by univariable analysis (p <0.004 Bonferroni correction) in a multivariable Cox In the final model, clinically relevant cut-off values were also used. Differences between Kaplan Meier event-free survival curves according to the predefined cut-off values were assessed by the log rank test.

All statistical tests were two-tailed. Data were analyzed using SPSS 10 (Microsoft ®) .

Results

Clinical features and laboratory findings of our population are described in Table 1.

Median age was 75 [72, 79] years. LV function was mildly reduced in this series: PLVEF was found in 131 patients (59%). EVR was present in 123 out of 220 patients (56 %).. During a median follow-up of 19 [START_REF] Arena | Peak VO2 and VE/VCO2 slope in patients with heart failure: a prognostic comparison[END_REF]40] months, 68 patients were admitted at least once for worsening HF (45) arrhythmias [START_REF] Scardovi | Cardiopulmonary exercise test is safe and reliable in elderly patients with chronic heart failure[END_REF] or acute coronary syndromes [START_REF] Guazzi | Cardiopulmonary exercise testing in the clinical and prognostic assessment of diastolic heart failure[END_REF] , and 32 died (17 of progressive HF, 9 of sudden death, 4 of other cardiovascular causes and 2 of cancer).

Among patients with left bundle branch 12 ( 5,4 % ) underwent to resincronization therapy.

Using a time-to first-event approach , overall 94 patients (43%) achieved the study end point of death or hospital admission. The significant predictors of major events by univariable analysis (p value < 0.004 using Bonferroni correction, Table 2) were entered in multiple multivariable Cox proportional hazard models in a sequential approach. Hazard ratios and related 95% CI per 10-unit increments and according to clinically relevant cut off values are shown in Table 3. Survival rates free from HF admission by the Kaplan Meier method according to renal dysfunction, BNP levels and VE/VCO2 slope are shown in Figures 234.

Discussion

The present prospective study is among the largest carried out to define, in a multimodal assessment, the additive prognostic role of CPET in an elderly population with mild to moderate HF and depressed or preserved LV function. Our data demonstrate that, in the "robust" elderly with HF able to perform maximal exercise, CPET is a crucial step in prognostic assessment and ventilatory parameters, together with evidence of renal dysfunction, have a key role in risk stratification over and beyond the level of LV function.

In advanced age morbidity, with its attendant impact on quality of life, is a relevant target for HF management beyond extension of life, so we chose to assess outcome in terms of hospital admissions for major cardiac events besides mortality: 43 % of the patients met, over a median period of 19 months, the combined end point.

Renal dysfunction, an established risk factor for adverse outcome in HF [START_REF] Mcalister | Renal insufficiency and heart failure: prognostic and therapeutic implications from a prospective cohort study[END_REF][START_REF] Hillege | Renal function as a predictor of outcome in a broad spectrum of patients with heart failure[END_REF], was in our elderly population a main independent prognostic predictor across the spectrum of ventricular impairment. This finding has important clinical implications: pharmacological management, focused on kidney protection to prevent or slow progression of renal impairment, may prove valuable also to improve outcome in elderly HF patients.

Our data further underscore the relevance of PLVEF-HF in the elderly: although LV function predicted outcome by univariable analysis, both renal dysfunction and BNP, a showed a stronger independent association with prognosis. Actually, the role of LVEF is far less important when morbidity, with its attending health care costs and detrimental impact on quality of life, is considered: in overt HF event-free survival does not differ among patients with depressed or preserved LV function [START_REF] Hogg | Heart failure with preserved left ventricular systolic function ; epidemiology, clinical characteristics and prognosis[END_REF][START_REF] Permenkil | Course and prognosis in patients > 70 years of age with congestive heart failure and normal versus abnormal left ventricular ejection fraction[END_REF][START_REF] Smith | Outcomes in heart failure patients with preserved ejection fraction. Mortality, readmission and functional decline[END_REF][START_REF] Ahmed | Outcomes in ambulatory chronic systolic and diastolic heart failure: a propensity score analysis[END_REF]. Moreover a recent clinical trial, which enrolled 556 HF patients with both preserved (EF> 50%) or reduced systolic function (EF < 50%), [START_REF] Ezekowitz | Comparison of one-year outcome (death and rehospitalization) in hospitalized heart failure patients with left ventricular ejection fraction >50% versus those with ejection fraction <50%[END_REF] demonstrated that PLVEF-HF presented a higher 6 months mortality rate than HF patients with depressed ejection fraction. These findings are also supported by another recent retrospective study [START_REF] Fonarow | OPTIMIZE-HF Investigators and Hospitals Characteristics, treatments, and outcomes of patients with preserved systolic function hospitalized for heart failure: a report from the OPTIMIZE-HF Registry[END_REF], that showed a similar post-discharge mortality and hospitalization rate in patients with preserved (EF >40%) and reduced (EF< 40%). The heterogeneous etiology, the high prevalence in the older population and the under-use of the optimal pharmacological treatment are probably the reason of the high morbidity and mortality rate of patients with PLVEF-HF and of the reduced prognostic significance of EF in this group of patients.

Our main aim in this study was to assess the additive prognostic role of CPET, an established test for risk stratification in other clinical HF settings, in elderly patients, who so far have been substantially neglected in the literature. Controversy still exists on the best ventilatory prognostic predictor in general [START_REF] Guazzi | Cardiopulmonary exercise testing in the clinical and prognostic assessment of diastolic heart failure[END_REF]. Peak exercise VO2 has been found superior in large series [START_REF] O'neill | Peak oxygen consumption as a predictor of death in patients with heart failure receiving beta-blockers[END_REF] and recent studies [START_REF] Ingle | Combining the ventilatory response to exercise and peak oxygen consumption is no better than peak oxygen consumption alone in predicting mortality in chronic heart failure[END_REF], and the anaerobic threshold in other studies of younger HF patients [START_REF] Gitt | Exercise anaerobic threshold and ventilatory efficiency identify heart failure patients for high risk of early death[END_REF]. Other reports stressed the optimal value of ventilatory efficiency as marker of adverse outcome [START_REF] Arena | Peak VO2 and VE/VCO2 slope in patients with heart failure: a prognostic comparison[END_REF][START_REF] Guazzi | Cardiopulmonary exercise testing in the clinical and prognostic assessment of diastolic heart failure[END_REF][START_REF] Scardovi | Cardiopulmonary exercise test is safe and reliable in elderly patients with chronic heart failure[END_REF][START_REF] Arena | The minute ventilation/carbon dioxide production slope is prognostically superior to the oxygen uptake efficiency slope[END_REF]. Old age per se might well determine differences in the definition of the most useful CPET parameters for outcome assessment. Peak exercise VO2 is known to be influenced, besides age and gender, by comorbidities and noncardiac factors, such as muscle deconditioning, motivation, anemia, all highly prevalent in the elderly, and might therefore be less useful as prognostic marker in this population, as highlighted by our A c c e p t e d M a n u s c r i p t findings. VO2AT, the anaerobic threshold, an objective parameter of cardiopulmonary exercise capacity that can be derived from submaximal exercise testing, previously found useful to predict 6-month mortality [START_REF] Gitt | Exercise anaerobic threshold and ventilatory efficiency identify heart failure patients for high risk of early death[END_REF], could not be determined in about 15% of our study patients and was not independently associated to prognosis in this group. So far, only three studies have analysed the prognostic value of CPET in patients of an age similar to ours [START_REF] Davies | Chronic heart failure in the elderly: value of cardiopulmonary exercise testing in risk stratification[END_REF][START_REF] Mejhert | Ventilarory variables are strong prognostic markers in elderly patients with heart failure[END_REF][START_REF] Cicoira | Chronic heart failure in the very elderly: clinical status, survival,and prognostic factors in 188 patients more than 70 years old[END_REF] . Both Davies et al [START_REF] Davies | Chronic heart failure in the elderly: value of cardiopulmonary exercise testing in risk stratification[END_REF] and Mejhert et al. [START_REF] Mejhert | Ventilarory variables are strong prognostic markers in elderly patients with heart failure[END_REF] demonstrated in small series the prominent predictive role of VE/VCO 2 slope, but their analyses were restricted to patients with SHF. In a larger study by Cicoira et al [START_REF] Cicoira | Chronic heart failure in the very elderly: clinical status, survival,and prognostic factors in 188 patients more than 70 years old[END_REF], among 102 SHF or PLVEF-HF patients with a maximal CPET (RER > 1) both PVO2 and VE/VCO2 slope were mortality predictors. The inclusion of subjects with hypertrophic or restrictive cardiomyopathy and dynamic flow obstruction, the different end point (death vs mortality and morbidity), and the underuse of beta-blockers (15% vs 56%) may explain the different prognostic weight of LVEF in Cicoira's and our experience. In none of these studies was CPET compared to BNP as a risk stratification tool.

In a group of patients with SHF and a median age of 71 years, we previously demonstrated the prognostic superiority of ventilatory and diastolic function parameters in comparison with BNP [START_REF] Scardovi | Multiparametric risk stratification in patients with mild to moderate chronic heart failure[END_REF]. In the present series, although independently predictive in the clinical model, as anticipated by its significant relationship with ventilatory parameters, BNP lost its power when ventilatory parameters, namely VE/VCO2 slope, were included in the multivariable model. Based on these findings, when CPET cannot be adequately performed in the elderly, BNP may be used as reliable surrogate predictor of prognosis.

The prognosis of elderly pts with CHF is poor, and NYHA classification, a simple and widely-used approach to the assessment of their functional status, seems unreliable for risk stratification. A c c e p t e d M a n u s c r i p t CPX, an inexpensive and reliable tool for testing cardio-pulmonary function can be safely performed in the vast majority of older patients, adding important information on disease severity and thus providing a guide for a tailored CHF treatment also in this population normally neglected by exercise test guidelines.

Study limitations

Our study population was relatively small and included only clinically stable "robust" outpatients with mild to moderate HF, who did not have important comorbidities and were furthermore able to perform a CPET. Therefore our conclusions cannot be extended to all elderly HF patients.

Beta-blockers reduced mortality and hospital admissions in the landmark HF trials, but did not predict outcome in the present series. One possible explanation is the relatively low proportion of sudden deaths with respect to worsening HF in our patients. Furthermore stratification of ventilatory parameters by beta-blocker treatment did not show significant differences.

We did not measure plasma N-terminal pro-BNP, recently presented as an accurate prognostic predictor in HF, as point-of-care testing, easier to perform, was not yet available for NT-pro-BNP. at the time the study was performed. Patients with advanced renal dysfunction were excluded from the study because of the non-specific elevation of plasma BNP in this clinical condition However, even in this relatively healthy population, mild degrees of renal dysfunction were one of the strongest predictors of outcome independently of BNP levels.

We did not assess the value of other emerging ventilatory parameters such as the oxygen uptake efficiency slope [START_REF] Davies | Enhanced prognostic value from cardiopulmonary exercise testing in chronic heart failure by non-linear analysis: oxygen uptake efficiency slope[END_REF]. Indeed others [START_REF] Arena | The minute ventilation/carbon dioxide production slope is prognostically superior to the oxygen uptake efficiency slope[END_REF] have recently reasserted the superior prognostic characteristics of the VE/VCO2 slope over both peak VO2, both at maximal and submaximal testing, in younger HF patients treated by beta-blockers in a proportion similar to our series.

Conclusion

In robust elderly patients with mild to moderate congestive HF and impaired or PLVEF, reduced estimated glomerular filtration rate and a steeper VE/VCO2 slope provide additional independent prognostic information across the spectrum of ventricular function and identify a high risk population commonly not considered in exercise test guidelines. These findings authorize to extend some clinical applications of CPX validated in middle-aged pts to older populations with CHF not eligible for heart transplantation suggesting a wider use of this technique. A c c e p t e d M a n u s c r i p t

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Clinical

  Science Immediate Publication. Published on 12 Aug 2008 as manuscript CS20080111 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080111

  proportional hazards model. in sequential diagnostic blocks of clinical and laboratory, 7 Clinical Science Immediate Publication. Published on 12 Aug 2008 as manuscript CS20080111 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080111 A c c e p t e d M a n u s c r i p t echocardiographic and CPET information, to mimick the sequence of information obtained in clinical practice. Relevant covariates were selected through a forward selection procedure. Hazard ratios (HR) are presented with 95% confidence intervals (CI). Non categorical variables were entered as continuous values and HR are expressed as risk per 10-unit change.

8

  Clinical Science Immediate Publication. Published on 12 Aug 2008 as manuscript CS20080111 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080111 A c c e p t e d M a n u s c r i p t In the first model, among 5 clinical, laboratory and echocardiographic variables, only renal dysfunction and higher BNP levels were predictive of the end point. In the final model including 4 CPET parameters, only renal dysfunction and a steeper VE/VCO2 slope were significantly associated to outcome.

9

  Clinical Science Immediate Publication. Published on 12 Aug 2008 as manuscript CS20080111 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080111 A c c e p t e d M a n u s c r i p t composite marker of systolic and diastolic function and neurohormonal activation [13],

11 Clinical

 11 Science Immediate Publication. Published on 12 Aug 2008 as manuscript CS20080111 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080111

Clinical

  Science Immediate Publication. Published on 12 Aug 2008 as manuscript CS20080111 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080111 A c c e p t e d M a n u s c r i p t Clinical Science Immediate Publication. Published on 12 Aug 2008 as manuscript CS20080111 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080111 A c c e p t e d M a n u s c r i p t

Fig. 1 Fig. 2 Fig. 3 Fig. 4

 1234 Fig.1 Study flow chart detailing exclusion criteria

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  Clinical Science Immediate Publication. Published on 12 Aug 2008 as manuscript CS20080111 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080111

Table 1

 1 Clinical characteristics and main parameters in the 220 study patients Clinical Science Immediate Publication. Published on 12 Aug 2008 as manuscript CS20080111 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080111 A c c e p t e d M a n u s c r i p t

	Clinical characteristics	N (%)		Median [Interquartile range]
	Female gender	70 (32 %)	Laboratory findings
	Ischemic etiology of CHF	101 (46%)	Hemoglobin ( g/ dl )	12.7 [11.7, 13.7]
	NYHA functional class III	51 (23 %)	Serum Sodium (mmol/l)	140 [138, 142]
	Left bundle branch block	62 (28%)	Creatinine cl. (ml/min )	50 [38, 63]
	Chronic atrial fibrillation	33 (15%)	BNP pg / ml	141 [56, 365]
	Diabetes	68 (31 %)
	Anamnestic mild BPCO	51 ( 23%)
	ACE-inhibitors	196 (89%)	Echocardiographic variables
	ARB	18 8%)	LVED volume (ml)	129 [103, 160]
	Beta-Blockers	123 (56%)	LVES volume (ml)	70 [50, 105]
	Non K + sparing diuretics 194 (88%)	LVEF (%)	42 [34, 51]
	K + sparing diuretics	53 (24%)	E deceleration time (msec)	200 [150, 225]
	Digitalis	13 (6%)	PAP (mmHg)	31 [25, 44]
	Nitrates	123 (56%)	Restrictive filling pattern	40 (22%)
	Oral anticoagulants	31 (14%)	Cardiopulmonary exercise test parameters
	CRT	5 (2%)	PVO 2 (ml/kg/min)	11.9 [9.76, 14]
	ICD	5 (2%)	% PVO 2 (%)	63 [51, 74]
			VO2AT(n=185)(ml/kg/min)	9.20 [7.8, 10.5]
			VE/VO 2	42 [37, 50]
			VE/VCO 2	37 [34, 43]
			VE/VCO 2 slope	33.2 [29, 38.1]
			VCO 2 slope/ PVO 2 ratio	2.78 [2.24, 3.72]
			Workload ( Watt )	61 [48, 77]

ACE, angiotensin converting enzyme ; ARB, angiotensin receptor blocking; CRT, cardiac resynchronization therapy; ICD, implantable cardioverter defibrillator; LVEF, left ventricular ejection fraction; LVED, left ventricular end diastolic; LVES, left ventricular end systolic; PAP, pulmonary artery systolic pressure; PVO 2 , peak oxygen uptake; % PVO 2, percent predicted PVO 2; VE, ventilation; VE/VCO 2 , relation between minute ventilation and carbon dioxide production;VE/VCO 2 slope, slope of the regression line relating VE to carbon dioxide production; VE/VO 2 , relation between minute ventilation and expired oxygen; VO2AT, anaerobic threshold.

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Table 2

 2 Univariable predictors of outcome

	p	HR	95% CI

Table 3

 3 Multivariable predictors of death or hospitalizationClinical Science Immediate Publication. Published on 12 Aug 2008 as manuscript CS20080111 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080111

	p	HR	95% CI

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Clinical Science Immediate Publication. Published on 12 Aug 2008 as manuscript CS20080111 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080111 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgments

The corresponding author affirms that she has listed everyone who contributed significantly to the work. None of the authors has any conflict of interest, such as financial or personal conflicts; moreover no circumstance or competing interest that could be construed or perceived as influencing the interpretation of the results occured. 13 Clinical Science Immediate Publication. Published on 12 Aug 2008 as manuscript CS20080111 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080111

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.