Ucn2 (urocortin 2) is a recently discovered peptide with therapeutic potential in heart failure. As any new treatment is likely to be used in conjunction with standard ACEI (angiotensin-converting enzyme inhibitor) therapy, it is important that the combined effects of these agents are assessed. In the present study, we investigated the effects of Ucn2 and an ACEI (captopril) administered for 3 h, both separately and together, in eight sheep with pacing-induced heart failure. Ucn2 and captopril alone both increased CO (cardiac output; Ucn2>captopril) and decreased arterial pressure (captopril>Ucn2), left atrial pressure (Ucn2>captopril) and peripheral resistance (Ucn2=captopril) relative to controls. Compared with either treatment alone, combined treatment further improved CO and reduced peripheral resistance and cardiac preload, without inducing further falls in blood pressure. In contrast with the marked increase in plasma renin activity observed with captopril alone, Ucn2 administration reduced renin activity, whereas the combined agents resulted in intermediate renin levels. All active treatments decreased circulating levels of aldosterone (Ucn2+captopril>Ucn2=captopril), endothelin-1 and the natriuretic peptides (Ucn2+captopril=Ucn2>captopril), whereas adrenaline (epinephrine) fell only with Ucn2 (Ucn2+captopril=Ucn2), and vasopressin increased during captopril alone. Ucn2, both separately and in conjunction with captopril, increased urine output, sodium and creatinine excretion and creatinine clearance. Conversely, captopril administered alone adversely affected these renal indices. In conclusion, co-treatment with Ucn2 and an ACEI in heart failure produced significantly greater improvements in haemodynamics, hormonal profile and renal function than achieved by captopril alone. These results indicate that dual treatment with these two agents is beneficial.

INTRODUCTION

Ucn2 (urocortin 2) is a recently identified member of the CRF (corticotropin-releasing factor) peptide family that binds selectively to the G-protein-coupled CRF receptor subtype CRF2 [1]. Both ligand and receptor are widely distributed throughout the brain and peripheral tissues, with a strong expression demonstrated in the heart and vasculature [2]. A range of biological actions have been reported for Ucn2 since its discovery, including vasodilator [2] and positive cardiac contractile effects in normal animals and humans [3,4], as well as cardioprotective actions against ischaemia/reperfusion injury in both mouse cardiomyocytes and the intact heart [5]. These findings suggest not only a role for the peptide in the regulation of cardiovascular function, but a possible involvement in the pathophysiology of cardiovascular disease. Administration of the peptide in a murine model of HF (heart failure) has been shown to induce inotropic and lusitropic actions and to improve cardiac performance [3]. In sheep with experimental HF, our group has demonstrated that Ucn2 produces significant reductions in peripheral resistance, arterial pressure and LAP (left atrial pressure) and increases in CO (cardiac output), in association with marked attenuation of a number of vasoconstrictor/volume-retaining systems [including the RAS (renin–angiotensin system), aldosterone, ET-1 (endothelin-1), AVP (arginine vasopressin) and adrenaline (epinephrine)] and augmentation of renal function [6]. More recently, we have shown that infusion of Ucn2 in humans with mild HF causes dose-related increases in ejection fraction and decreases in vascular resistance [7].

Although these studies indicate a therapeutic potential of Ucn2 in HF, any new treatment is likely to be used in conjunction with standard ACEI [ACE (angiotensin-converting enzyme) inhibitor] therapy, making it important that the combined effects of these agents are assessed. This is of particular interest given the interactions we have observed previously between Ucn2 and the RAS and aldosterone [6,8], and conflicting reports of an involvement of the RAS in the vasodilatory activity of the Ucn peptides [9,10].

In the present study, we investigated for the first time the haemodynamic, hormonal and renal effects of Ucn2 and an ACEI (captopril) administered both separately and together in sheep with experimental HF.

MATERIALS AND METHODS

Surgical preparation of sheep

Eight Coopworth ewes (50–59 kg) were instrumented via a left lateral thoracotomy under general anaesthesia (induced by 17 mg of thiopentone/kg of body weight; maintained with halothane/nitrous oxide) as described previously [11]. Briefly, two polyvinyl chloride catheters were inserted into the left atrium for blood sampling and LAP determination, a Konigsberg pressure-tip transducer was inserted into the aorta to record MAP (mean arterial pressure) and into the apex of the left ventricle to obtain maximum derivatives of pressure over time (dP/dtmax) as an index of contractility, an electromagnetic flow probe was placed around the ascending aorta to measure CO, a Swan–Ganz catheter was inserted into the pulmonary artery for infusions, and a 7 French His-bundle electrode was stitched subepicardially to the wall of the left ventricle for pacing. A bladder catheter was inserted per urethra for urine collections. Animals recovered for at least 14 days before commencing the study protocol. During the experiments, the animals were held in metabolic cages and had free access to water and food (containing 80 mmol of sodium/day and 200 mmol of potassium/day).

Study protocol

HF was induced by 7 days of rapid LV (left ventricular) pacing at 225 beats/min [11,12] and was maintained by continuous pacing for the duration of the study. On four separate days (days 8, 10, 12 and 14 of pacing) with a washout day between each, the sheep received in a balanced random-order design a vehicle control (0.9% saline), murine Ucn2 (50 μg bolus plus an infusion at 50 μg/h; American Peptide Company), an ACEI (captopril; 15 mg bolus plus an infusion at 3 mg/h), and the combination of both agents. The study operator was not blinded to the treatment order. All treatments were administered for 3 h in a total volume of 50 ml via the pulmonary artery catheter. The doses of both Ucn2 and captopril chosen were based on our previous experience with these agents in the same animal model [6,13].

MAP, LAP, CO, dP/dtmax and CTPR [calculated TPR (total peripheral resistance); calculated as MAP/CO] were recorded at 15 min intervals in the hour prior to infusion (baseline), and at 15, 30, 45, 60, 90, 120, 150 and 180 min during both the 3 h infusion and post-infusion periods. Haemodynamic measurements were determined by online computer assisted analysis (PowerLab Systems; ADInstruments) using established methods [14].

Blood samples were drawn from the left atrium 30 min and immediately pre-infusion (baseline), and at 30, 60, 120 and 180 min during the 3 h infusion and post-infusion periods. Samples were taken into EDTA tubes on ice, centrifuged at 4 °C and stored at either −20 °C or −80 °C before assay for PRA (plasma renin activity), aldosterone, AVP, ET-1, ANP (atrial natriuretic peptide), BNP (brain natriuretic peptide) and catecholamines [11,1315]. For each hormone, all samples from individual animals were measured in the same assay to avoid inter-assay variability. Plasma electrolytes and haematocrit were measured with every blood sample taken.

Urine was collected hourly for the measurement of volume and sodium, potassium and creatinine excretion. Creatinine clearance was calculated as urine creatinine/plasma creatinine.

The study protocol was approved by the local Animal Ethics Committee.

Statistics

Results are expressed as means±S.E.M. Baseline haemodynamic and hormone values are the means of four and two measurements respectively, made within the hour immediately pre-infusion. Differences between non-paced laboratory normal sheep (n=20) and HF animals (vehicle control baseline data) were compared using independent Student's t tests (see Table 1). Differences between the four separate study arms (vehicle, Ucn2, captopril and Ucn2+captopril) were analysed by two-way repeated measures ANOVA using the SPSS statistical package (version 11.0.2). Significance was assumed when P<0.05. Where significant differences were identified by ANOVA, the level of significance at individual time points in Figure 4 and Table 2 was determined by Fisher's protected least-significant difference tests.

Table 1
Effects of rapid LV pacing

Values are means±S.E.M. from sheep before (non-paced; laboratory normal data, n=20) and after induction of HF by 7 days of rapid LV pacing at 225 beats/min (paced; vehicle control baseline data, n=8). **P<0.01 and †P<0.001 compared with non-paced animals.

 Non-paced Paced 
CO (litres/min) 4.2±0.4 2.2±0.4† 
dP/dtmax (mmHg/s) 2089±153 1161±106† 
MAP (mmHg) 83±2 73±2† 
LAP (mmHg) 4.1±0.3  23.9±1.0† 
TPR (mmHg·litre−1·min−120±3 40±6† 
ANP (pmol/l) 17±2 340±31† 
BNP (pmol/l) 3±1 59±5† 
PRA (nmol·litre−1·h−10.39±0.06 2.83±0.38† 
Aldosterone (pmol/l) 225±24 2855±595† 
ET-1 (pmol/l) 1.68±0.08 4.71±0.43† 
AVP (pmol/l) 1.7±0.1 2.3±0.8** 
Noradrenaline (pmol/l) 2683±507 6811±2024† 
Adrenaline (pmol/l) 490±88 942±95** 
Urine volume (ml/h) 81±11 74±20 
Urinary sodium excretion (mmol/h) 2.62±0.30 0.18±0.06† 
Urinary potassium excretion (mmol/h) 9.0±0.6 4.2±0.9** 
Urinary creatinine excretion (mmol/h) 0.50±0.02 0.35±0.02† 
Creatinine clearance (ml/min) 121±9 66±5† 
Haematocrit (%) 31.2±1.2 27.4±1.0** 
 Non-paced Paced 
CO (litres/min) 4.2±0.4 2.2±0.4† 
dP/dtmax (mmHg/s) 2089±153 1161±106† 
MAP (mmHg) 83±2 73±2† 
LAP (mmHg) 4.1±0.3  23.9±1.0† 
TPR (mmHg·litre−1·min−120±3 40±6† 
ANP (pmol/l) 17±2 340±31† 
BNP (pmol/l) 3±1 59±5† 
PRA (nmol·litre−1·h−10.39±0.06 2.83±0.38† 
Aldosterone (pmol/l) 225±24 2855±595† 
ET-1 (pmol/l) 1.68±0.08 4.71±0.43† 
AVP (pmol/l) 1.7±0.1 2.3±0.8** 
Noradrenaline (pmol/l) 2683±507 6811±2024† 
Adrenaline (pmol/l) 490±88 942±95** 
Urine volume (ml/h) 81±11 74±20 
Urinary sodium excretion (mmol/h) 2.62±0.30 0.18±0.06† 
Urinary potassium excretion (mmol/h) 9.0±0.6 4.2±0.9** 
Urinary creatinine excretion (mmol/h) 0.50±0.02 0.35±0.02† 
Creatinine clearance (ml/min) 121±9 66±5† 
Haematocrit (%) 31.2±1.2 27.4±1.0** 
Table 2
Effects of Ucn2 and captopril, separately and combined, in sheep with HF

Values are mean±S.E.M. responses to 3 h intravenous infusions of vehicle control, Ucn2, captopril and Ucn2+catopril in eight sheep with pacing-induced HF. *P<0.05 and †P<0.001 compared with time-matched vehicle controls.

  Treatment period   
Parameter Baseline 1 h 2 h 3 h 4 h 6 h 
dP/dtmax (mmHg/s)       
 Vehicle 1161±106 1166±109 1144±108 1138±107 1142±108 1128±105 
 Ucn2 1159±105 1864±135† 1949±190† 2043±220† 1654±154† 1272±109* 
 Captopril 1167±106 1132±122 1140±132 1137±125 1157±127 1130±104 
 Ucn2+captorpil 1145±107 1855±193† 1868±198† 1836±202† 1545±147† 1212±102 
Haematocrit (%)       
 Vehicle 27.4±1.0 26.4±1.0 26.0±1.0 26.1±1.0 25.7±1.2 24.9±1.1 
 Ucn2 26.8±0.9 24.3±1.1† 23.1±0.9† 23.2±0.6† 23.3±0.7† 24.2±0.8 
 Captopril 27.1±1.0 27.9±1.0† 28.8±1.3† 29.2±1.4† 28.7±1.5† 27.3±1.3 
 Ucn2+captorpil 26.8±0.8 26.4±0.8 26.7±0.6 26.3±0.7 25.1±0.7 24.9±0.6 
Plasma BNP (pmol/l)       
 Vehicle 58.8±4.7 61.5±10.5 65.0±11.5 70.4±13.7 65.8±7.9 62.9±9.6 
 Ucn2 70.3±8.5 28.9±4.3† 19.4±3.0† 20.4±3.5† 26.3±5.5† 33.1±7.5† 
 Captopril 59.3±5.8 42.6±4.0† 47.4±6.3† 45.6±3.8† 43.4±5.2† 35.8±3.8† 
 Ucn2+captorpil 74.8±8.7 32.1±4.2† 29.9±3.7† 22.5±2.8† 27.0±3.8† 32.2±5.9† 
Plasma noradrenaline (nmol/l)       
 Vehicle 6.8±2.0 13.3±4.1 8.3±2.3 6.8±1.7 8.9±2.5 11.7±4.7 
 Ucn2 5.8±1.7 8.0±1.8 4.9±1.0 5.1±1.1 6.5±1.0 7.8±1.7 
 Captopril 4.7±1.2 11.9±2.4 8.6±2.0 8.5±2.2 9.2±2.3 9.1±2.3 
 Ucn2+captorpil 4.4±0.8 8.6±1.6 7.2±1.9 6.8±1.5 8.2±1.6 8.6±1.9 
Plasma creatinine (μmol/l)       
 Vehicle 86.3±4.6 86.3±4.6 86.3±4.6 85.0±5.0 85.0±5.0 83.8±4.2 
 Ucn2 89.9±5.0 86.3±6.3 85.0±5.3 83.8±4.9* 83.8±5.0* 83.8±4.9 
 Captopril 85.0±5.3 93.8±5.0† 92.5±5.3† 91.3±5.8† 92.5±5.9† 90.0±7.8† 
 Ucn2+captorpil 88.8±4.8 87.5±4.5 85.0±5.3 81.3±4.8* 81.3±4.4* 82.5±4.1 
Creatinine clearance (ml/min)       
 Vehicle 66.4±4.6 73.0±4.7 75.8±5.7 70.3±2.6 67.8±4.1 64.3±3.6 
 Ucn2 66.0±4.6 93.9±11.6† 98.4±8.1† 96.9±9.7† 90.7±9.1† 79.2±4.7† 
 Captopril 68.5±4.1 37.1±11.7† 56.7±11.2† 57.3±11.0* 69.0±11.1 68.9±7.3 
 Ucn2+captorpil 63.5±2.5 84.6±11.3* 86.3±10.7* 96.7±6.0† 90.3±6.2† 78.9±3.7* 
  Treatment period   
Parameter Baseline 1 h 2 h 3 h 4 h 6 h 
dP/dtmax (mmHg/s)       
 Vehicle 1161±106 1166±109 1144±108 1138±107 1142±108 1128±105 
 Ucn2 1159±105 1864±135† 1949±190† 2043±220† 1654±154† 1272±109* 
 Captopril 1167±106 1132±122 1140±132 1137±125 1157±127 1130±104 
 Ucn2+captorpil 1145±107 1855±193† 1868±198† 1836±202† 1545±147† 1212±102 
Haematocrit (%)       
 Vehicle 27.4±1.0 26.4±1.0 26.0±1.0 26.1±1.0 25.7±1.2 24.9±1.1 
 Ucn2 26.8±0.9 24.3±1.1† 23.1±0.9† 23.2±0.6† 23.3±0.7† 24.2±0.8 
 Captopril 27.1±1.0 27.9±1.0† 28.8±1.3† 29.2±1.4† 28.7±1.5† 27.3±1.3 
 Ucn2+captorpil 26.8±0.8 26.4±0.8 26.7±0.6 26.3±0.7 25.1±0.7 24.9±0.6 
Plasma BNP (pmol/l)       
 Vehicle 58.8±4.7 61.5±10.5 65.0±11.5 70.4±13.7 65.8±7.9 62.9±9.6 
 Ucn2 70.3±8.5 28.9±4.3† 19.4±3.0† 20.4±3.5† 26.3±5.5† 33.1±7.5† 
 Captopril 59.3±5.8 42.6±4.0† 47.4±6.3† 45.6±3.8† 43.4±5.2† 35.8±3.8† 
 Ucn2+captorpil 74.8±8.7 32.1±4.2† 29.9±3.7† 22.5±2.8† 27.0±3.8† 32.2±5.9† 
Plasma noradrenaline (nmol/l)       
 Vehicle 6.8±2.0 13.3±4.1 8.3±2.3 6.8±1.7 8.9±2.5 11.7±4.7 
 Ucn2 5.8±1.7 8.0±1.8 4.9±1.0 5.1±1.1 6.5±1.0 7.8±1.7 
 Captopril 4.7±1.2 11.9±2.4 8.6±2.0 8.5±2.2 9.2±2.3 9.1±2.3 
 Ucn2+captorpil 4.4±0.8 8.6±1.6 7.2±1.9 6.8±1.5 8.2±1.6 8.6±1.9 
Plasma creatinine (μmol/l)       
 Vehicle 86.3±4.6 86.3±4.6 86.3±4.6 85.0±5.0 85.0±5.0 83.8±4.2 
 Ucn2 89.9±5.0 86.3±6.3 85.0±5.3 83.8±4.9* 83.8±5.0* 83.8±4.9 
 Captopril 85.0±5.3 93.8±5.0† 92.5±5.3† 91.3±5.8† 92.5±5.9† 90.0±7.8† 
 Ucn2+captorpil 88.8±4.8 87.5±4.5 85.0±5.3 81.3±4.8* 81.3±4.4* 82.5±4.1 
Creatinine clearance (ml/min)       
 Vehicle 66.4±4.6 73.0±4.7 75.8±5.7 70.3±2.6 67.8±4.1 64.3±3.6 
 Ucn2 66.0±4.6 93.9±11.6† 98.4±8.1† 96.9±9.7† 90.7±9.1† 79.2±4.7† 
 Captopril 68.5±4.1 37.1±11.7† 56.7±11.2† 57.3±11.0* 69.0±11.1 68.9±7.3 
 Ucn2+captorpil 63.5±2.5 84.6±11.3* 86.3±10.7* 96.7±6.0† 90.3±6.2† 78.9±3.7* 

RESULTS

Rapid LV pacing at 225 beats/min for 7 days produced the haemodynamic, endocrine and sodium-retaining hallmarks of congestive HF [10,11], with reduced CO, MAP and renal function, elevated LAP and peripheral resistance, and widespread hormonal activation (Table 1).

Compared with time-matched controls, separate Ucn2 and captopril administration significantly increased CO (Ucn2>captopril; both P<0.001) and reduced MAP (captopril>Ucn2; both P<0.001), LAP (Ucn2>captopril; both P<0.001) and CTPR (Ucn2=captopril; both P<0.001) (Figure 1). Only Ucn2 and combined Ucn2+captopril induced an increase in dP/dtmax (Ucn2=Ucn2+captopril; both P<0.001) (Table 2). Haematocrit was elevated by captopril alone (P<0.001), decreased by Ucn2 (P<0.01), and was unchanged during dual treatment (Table 2). Compared with either agent separately, combined Ucn2+captopril further improved CO (P<0.001 compared with both agents alone), and reduced peripheral resistance (P<0.001 compared with both agents alone) and cardiac preload (P<0.001 compared with both agents alone) (Figure 1).

Haemodynamic responses to 3 h infusions of vehicle, Ucn2, captopril and Ucn2+captopril in eight sheep with HF

Figure 1
Haemodynamic responses to 3 h infusions of vehicle, Ucn2, captopril and Ucn2+captopril in eight sheep with HF

Values are means±S.E.M. Ucn2 was given as a 50 μg bolus and an infusion at 50 μg/h; captopril was given as a 15 mg bolus and an infusion at 3 mg/h.

Figure 1
Haemodynamic responses to 3 h infusions of vehicle, Ucn2, captopril and Ucn2+captopril in eight sheep with HF

Values are means±S.E.M. Ucn2 was given as a 50 μg bolus and an infusion at 50 μg/h; captopril was given as a 15 mg bolus and an infusion at 3 mg/h.

In contrast with the marked increase in PRA observed during captopril alone (P<0.001), Ucn2 administration reduced PRA compared with control (P<0.01) (Figure 2). Although PRA was also elevated by the combined agents (P<0.001), levels were appreciably lower than those seen with ACE inhibition alone (P<0.01). On the other hand, plasma aldosterone was significantly decreased by all treatments (P<0.001 for Ucn2; P<0.01 for captopril; P<0.001 for Ucn2+captopril), with reductions during Ucn2+captopril significantly greater than achieved by either drug individually (P<0.05 compared with Ucn2 alone; P<0.001 compared with captopril alone) (Figure 2).

PRA and aldosterone responses to 3 h infusions of vehicle, Ucn2, captopril and Ucn2+captopril in eight sheep with HF

Figure 2
PRA and aldosterone responses to 3 h infusions of vehicle, Ucn2, captopril and Ucn2+captopril in eight sheep with HF

Values are means±S.E.M. Ucn2 was given as a 50 μg bolus and an infusion at 50 μg/h; catopril was given as a 15 mg bolus and an infusion at 3 mg/h.

Figure 2
PRA and aldosterone responses to 3 h infusions of vehicle, Ucn2, captopril and Ucn2+captopril in eight sheep with HF

Values are means±S.E.M. Ucn2 was given as a 50 μg bolus and an infusion at 50 μg/h; catopril was given as a 15 mg bolus and an infusion at 3 mg/h.

All active treatments reduced circulating levels of ET-1 (all P<0.05), as well as ANP (P<0.001 for Ucn2 and Ucn2+captopril; P<0.05 for captopril) (Figure 3) and BNP (P<0.001 for Ucn2 and Ucn2+captopril; P<0.01 for captopril (Table 2). Plasma AVP rose slightly, but significantly, during treatment with captopril alone (P<0.05) and was unchanged relative to control by either Ucn2 or Ucn2+captopril (Figure 3) (although concentrations tended to decline post-treatment), and adrenaline levels fell during Ucn2 and Ucn2+captopril (both P<0.05) and rose post-captopril administration (P<0.01) (Figure 3). Plasma noradrenaline was unaltered by any agent (Table 2). Although the hormonal responses to Ucn2 and Ucn2+captopril treatments did not differ, except in the case of the natriuretic peptides, which tended to be reduced further by separate Ucn2 (P<0.05 for ANP; not significant for BNP), the combination of drugs produced significantly greater decreases in ET-1 (P<0.001), the natriuretic peptides (both P<0.001), AVP (P<0.01) and adrenaline (P<0.05) compared with captopril alone.

Hormonal responses to 3 h infusions of vehicle, Ucn2, captopril and Ucn2+captopril in eight sheep with HF

Figure 3
Hormonal responses to 3 h infusions of vehicle, Ucn2, captopril and Ucn2+captopril in eight sheep with HF

Values are means±S.E.M. Ucn2 was given as a 50 μg bolus and an infusion at 50 μg/h; catopril was given as a 15 mg bolus and an infusion at 3 mg/h.

Figure 3
Hormonal responses to 3 h infusions of vehicle, Ucn2, captopril and Ucn2+captopril in eight sheep with HF

Values are means±S.E.M. Ucn2 was given as a 50 μg bolus and an infusion at 50 μg/h; catopril was given as a 15 mg bolus and an infusion at 3 mg/h.

Ucn2, both separately and in conjunction with captopril, increased urine output (both P<0.05), urine sodium (P<0.01 for Ucn2; P<0.05 for Ucn2+captopril), potassium (P<0.001 for Ucn2; P<0.05 for Ucn2+captopril) and creatinine excretion (P<0.001 for Ucn2; P<0.01 for Ucn2+captopril) (Figure 4) as well as creatinine clearance (Ucn2, P<0.001; Ucn2+captopril, P<0.05) (Table 2). Conversely, captopril administered alone resulted in decreases in these renal indices relative to controls (P<0.05 for urine sodium; P<0.001 for urine creatinine; P<0.001 for creatinine clearance). Although renal responses were not significantly different between Ucn2+captopril and Ucn2, the effects of Ucn2+captopril and captopril alone contrasted markedly (P<0.05 for urine volume; P<0.01 for sodium; P<0.01 for potassium; P<0.001 for creatinine; P<0.01 for creatinine clearance).

Renal responses to 3 h infusions of vehicle, Ucn2, captopril and Ucn2+captopril in eight sheep with HF

Figure 4
Renal responses to 3 h infusions of vehicle, Ucn2, captopril and Ucn2+captopril in eight sheep with HF

Values are means±S.E.M. Ucn2 was given as a 50 μg bolus and an infusion at 50 μg/h; catopril was given as a 15 mg bolus and an infusion at 3 mg/h. *P<0.05, **P<0.01 and †P<0.001 compared with time-matched controls.

Figure 4
Renal responses to 3 h infusions of vehicle, Ucn2, captopril and Ucn2+captopril in eight sheep with HF

Values are means±S.E.M. Ucn2 was given as a 50 μg bolus and an infusion at 50 μg/h; catopril was given as a 15 mg bolus and an infusion at 3 mg/h. *P<0.05, **P<0.01 and †P<0.001 compared with time-matched controls.

Plasma creatinine levels, compared with controls, were increased by captopril and reduced by Ucn2 and Ucn2+captopril (all P<0.05) (Table 2). Compared with controls (3.9±0.1 mmol/l), potassium concentrations were also reduced by Ucn2 (3.7±0.2 mmol/l; P<0.01) and Ucn2+captopril (3.7±0.1 mmol/l; P<0.01) administration, but were unchanged during captopril (4.0±0.2 mmol/l) administration. Plasma sodium was unaltered by any treatment (results not shown).

DISCUSSION

The present study is the first to investigate the integrated effects of combined Ucn2 and ACE inhibition in HF in sheep, with results indicating that co-treatment has beneficial effects on haemodynamic, hormonal and renal indices in this disease beyond those seen with either treatment alone.

We found that Ucn2 and captopril substantially and similarly reduced CTPR (by approx. 40%), suggesting actions of both agents to reduce arteriolar tone. Although ACE inhibition acts primarily via mitigation of AngII (angiotensin II)-mediated vasoconstriction, Ucn2 is reported to act largely through direct stimulation of its CRF2 receptors in the vasculature [2,16], although effects of the peptide to reduce circulating levels of the vasoconstrictors AngII (indicated in the present study by falls in PRA), ET-1, AVP and adrenaline (all elevated in this model of HF), and reverse ET-1-induced arterial constriction [2], are also likely to have contributed to its modulation of vascular tone. Direct involvement of the RAS in Ucn-induced vasodilation has been suggested in a recent study by Yang et al. [10], reporting that the peptide dose-dependently reduces serum ACE activity (and therefore presumably AngII levels) in rats. On the other hand, Gardiner et al. [9] found that the effect of Ucn2 to elevate vascular conductance in a number of rat tissue beds was unaltered in the presence of the angiotensin receptor antagonist losartan, although it should be noted that this study was performed in normal animals where the RAS was not activated. Although the present study does not address the possibility of direct involvement or otherwise of the RAS in Ucn2-induced effects on arterial tone, we found combined treatment with Ucn2 and captopril augmented the decrease in CTPR by an additional 20% compared with either agent alone (normalizing peripheral resistance to levels observed in normal sheep), perhaps suggesting any RAS involvement to be minimal.

Although the actions of Ucn2 and captopril to reduce CTPR were comparable, their hypotensive effects were far from identical, with decreases in MAP induced by ACE inhibition (and equally by the combined treatments) markedly greater than that produced by Ucn2 alone, which is likely to be attributed to the considerably greater increase in CO seen with Ucn2. From a therapeutic viewpoint, the combination of Ucn2 and an ACEI (resulting in additional decreases in peripheral resistance without further concomitant reductions in blood pressure) could be attractive in this regard since symptomatic hypotension is a known complication of ACEI therapy, especially in severe grades of HF where blood flow to vital organs (such as the kidney) may be compromised [17]. Although the large decreases in cardiac afterload (peripheral resistance) probably contributed to the increase in CO associated with both treatments, the greater increase seen with Ucn2 presumably reflects the potent inotropic activity of the peptide, as suggested by the concurrent increase in dP/dt. This concurs with in vitro studies demonstrating that Ucn2 improves contractility in isolated mouse cardiomyocytes [18]. Combined Ucn2+captopril administration induced a small supplementary increase in cardiac performance relative to that produced by Ucn2 alone, and, although this additional increase might not be considered favourable in terms of possible elevated myocardial oxygen demand, the auxiliary actions of the Ucns to dilate coronary arteries [19], improve cardiac bioenergetics (through preservation of high-energy phosphate stores) [20] and cardiomyocyte intracellular calcium handling [18], and to lessen cardiac workload through additional reductions in peripheral resistance, may counterbalance any adverse consequence. The cardioprotective [5] and anti-arrhythmic activities [21] demonstrated by the Ucns contribute further to their beneficial cardiac effects.

Decreases in LAP observed with both treatments are likely to reflect, to a large degree, the increases in CO, with the greater Ucn2-induced decreases in cardiac pre-load associated with greater CO augmentation, although the lusitropic actions of this peptide [3], and possible venodilator activity to reduce circulatory filling pressures (as demonstrated previously for Ucn1) [22], may also have contributed. Co-treatment resulted in additional significant reductions in LAP to near normal levels. The haemodynamic responses observed in the present study to separate Ucn2 administration and ACE inhibition are identical with those we have demonstrated previously in this ovine model of HF [6,13], with our present results showing that the combination of these two agents produces a more beneficial haemodynamic profile than that achieved by either treatment alone.

A significant difference between Ucn2 and ACE inhibition in the present study concerns the response of PRA. Whereas captopril induced a prominent increase in PRA, a well-established consequence of ACE inhibition [23], Ucn2 decreased PRA. This occurred despite a decrease in blood pressure (albeit to a far lesser degree than that produced by captopril), but whether as a result of direct inhibition of renin release, increased sodium (and chloride) delivery to the macula densa (evidenced by the significant natriuresis), a decline in sympathetic drive to the juxtaglomerular cells or some other renin-inhibitory mechanism is unknown. The combined treatments produced an intermediate increase in PRA, approx. 66% of that seen with captopril administration alone, in the face of similar blood pressure reductions (and presumably a similar decrease in circulating AngII), although co-treatment was also associated with a significant (renin-inhibitory) increase in sodium excretion. Whatever the mechanism(s), the suppression by Ucn2 of the stimulatory action of ACE inhibition on PRA must be seen as favourable, with an additional possibility of long-term benefits since renin (and prorenin), through specific receptors [24], may have adverse effects via, for example, stimulation of profibrotic proteins [25].

Both treatments individually were associated with similar and marked reductions in plasma aldosterone levels, with the Ucn2-induced response being of slower onset yet more sustained. In the case of Ucn2, reductions in aldosterone levels presumably reflect decreased circulating AngII, as assessed by the decrease in PRA (and perhaps plasma potassium levels), although it is possible the peptide also has a direct inhibitory effect on aldosterone secretion [1]. The combined agents produced an even greater reduction in aldosterone levels that was prolonged beyond the treatment period as with Ucn2 alone. Such a pronounced aldosterone-inhibitory action of the two treatments together could have beneficial clinical effects given the robust evidence that aldosterone has adverse effects in patients with HF, even those receiving ACEIs [26].

Although plasma ET-1 levels were decreased with all of the active treatments, reductions were significantly greater with Ucn2 included. Circulating adrenaline was likewise decreased in the presence of Ucn2 (with levels actually rising above control following cessation of separate captopril administration). In addition, the peptide prevented the increase in AVP observed with ACE inhibition alone. Although the attenuation of these constrictor peptides by Ucn2 corresponds with previous findings in sheep with HF [6], the mechanisms behind these responses are yet to be investigated. It is unlikely, however, that these changes merely reflect an improvement in haemodynamic status (or function) given the disparate responses evident with separate captopril administration (which was also associated with haemodynamic recovery). Our present findings indicate an overall benefit of Ucn2 co-treatment on the suppression of vasoconstricting growth-promoting hormones in the setting of HF.

Both Ucn2 and captopril significantly reduced plasma levels of the natriuretic peptides (Ucn2>captopril), findings that have been reported previously for both ACE inhibition [13] and Ucn2 administration [6] in HF, and are presumably a consequence of the respective decreases in cardiac transmural distending pressures leading to reduced stimulus for secretion. Surprisingly, the combined treatment, which produced a significantly greater decline in LAP than that achieved with Ucn2 alone, was associated with smaller decreases in ANP and BNP than those seen with Ucn2. This may reflect the larger decreases in plasma levels of AngII (as judged by reductions in aldosterone), a potent secretagogue for natriuretic peptide secretion [27], with combined treatments.

In agreement with our previous study [6], Ucn2 increased urine output and urine sodium and creatinine excretion in these sheep with HF notwithstanding major decreases in circulating levels of the natriuretic peptides and a reduction in arterial pressure (and therefore renal perfusion pressure). Possible mechanisms contributing to these responses include improvements in glomerular filtration (assessed by the sizable increase in creatinine clearance), renal vasodilation [28], attenuation of antinatriuretic/antidiuretic factors (AngII, aldosterone and AVP) and perhaps direct tubular actions given the expression of Ucn2 within the kidney [1]. It is also conceivable that Ucn2 may stimulate the production of ANP/BNP within the kidney in view of reports demonstrating that the peptide augments natriuretic peptide secretion in cardiomyocytes [29]. In contrast, captopril administration, which was associated with major decreases in MAP, produced decreases in the renal indices measured, a finding in accordance with clinical studies showing that ACE inhibition in some patients results in a deterioration in renal function due to hypotension and reduced glomerular filtration at low perfusion pressures [30]. What is remarkable is that the combined treatment resulted in an improvement in renal performance (increases in urine volume, sodium and creatinine excretion and creatinine clearance) comparable with that generated in response to Ucn2 alone, despite decreases in blood pressure equal to captopril-induced reductions. Whatever the mechanisms underlying these renal effects, the ability of Ucn2 co-treatment with captopril to actually improve renal performance in the face of such low renal perfusion pressures, in a state already characterized by volume/sodium retention, could prove to be an important benefit in the clinical setting of HF.

In conclusion, co-treatment of Ucn2 with an ACEI in an experimental model of HF produced significantly greater improvements in cardiac performance and decreases in peripheral resistance and ventricular filling pressures than achieved by captopril alone, in association with further reductions in plasma aldosterone and ET-1. Ucn2 co-treatment also attenuated the captopril-induced increases in PRA, adrenaline and AVP and augmented renal function, despite decreases in blood pressure comparable with that induced by ACE inhibition by itself. These results indicate that dual treatment has multiple additive benefits in HF and should encourage longer-term studies of the co-administration of Ucn2 and ACE inhibition.

Abbreviations

     
  • ACE

    angiotensin-converting enzyme

  •  
  • ACEI

    ACE inhibitor

  •  
  • AngII

    angiotensin II

  •  
  • ANP

    atrial natriuretic peptide

  •  
  • AVP

    arginine vasopressin

  •  
  • BNP

    brain natriuretic peptide

  •  
  • CO

    cardiac output

  •  
  • CRF

    corticotropin-releasing factor

  •  
  • ET-1

    endothelin-1

  •  
  • HF

    heart failure

  •  
  • LAP

    left atrial pressure

  •  
  • LV

    left ventricular

  •  
  • MAP

    mean arterial pressure

  •  
  • PRA

    plasma renin activity

  •  
  • RAS

    renin–angiotensin system

  •  
  • TPR

    total peripheral resistance

  •  
  • CTPR

    calculated TPR

  •  
  • Ucn

    urocortin

We are grateful to The National Heart Foundation and Health Research Council of New Zealand for financial support, the New Zealand Lottery Grants Board for equipment, the staff of the Endocrine Laboratory for performing the hormone assays, and the staff of the Christchurch School of Medicine Animal Laboratory for animal care.

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