Christchurch Cardioendocrine Research Group, Department of Medicine, The Christchurch School of Medicine and Health Sciences, PO Box 4345, Christchurch, New Zealand
Received 7 October 2004; revised 30 March 2005; accepted 12 May 2005; online publish-ahead-of-print 16 June 2005.
* Corresponding author. Tel: +64 3 3640544; fax: +64 3 3640525. E-mail address: miriam.rademaker{at}chmeds.ac.nz
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Abstract |
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Methods and results In eight sheep with pacing-induced HF, a 4-day infusion of Ucn-I (0.3 µg/kg/h) induced prompt (30 min) and sustained (4-day) increases in cardiac output (CO, Day 4: 1.8±0.2 vs. 2.3±0.2 L/min, P<0.001) and stroke volume (7.8±0.8 vs. 10.2±1.0 mL/beat, P=0.0011), and reductions in mean arterial pressure (MAP, 72±3 vs. 70±3 mmHg, P=0.0305), left atrial pressure (26±1 vs. 11±2 mmHg, P<0.001), and total calculated peripheral resistance (43±6 vs. 32±4 mmHg/L/min, P<0.001). Ucn-I also induced persistent falls in plasma renin (1.34±0.23 vs. 0.77±0.10 nmol/L/min, P=0.048), aldosterone (3273±1172 vs. 382±44 pmol/L, P=0.0098), endothelin-1 (4.6±0.3 vs. 2.7±0.3 pmol/L, P<0.001), vasopressin (24±4 vs. 14±2 pmol/L, P=0.0028) and atrial (184±14 vs. 154±29 pmol/L, P=0.0226) and brain (43±5 vs. 32±6 pmol/L, P=0.0016) natriuretic peptides. Plasma adrenocorticotrophic hormone and cortisol rose transiently on Day 0. Ucn-I enhanced urinary sodium excretion (5.3-fold, P=0.0001) and creatinine clearance (1.3-fold, P=0.0055) long-term, and tended to increase urine output (P=0.0748). Food intake was attenuated over the first 2 days of treatment (P=0.0283).
Conclusion Four-day administration of Ucn-I induces sustained reductions in cardiac preload and MAP, improvements in CO and renal function, and inhibition of a range of vasoconstrictor/volume-retaining factors. These findings support Ucn-I's therapeutic potential in HF.
Key Words: Urocortin-I Heart failure Pharmacology Blood pressure Cardiac output Hormones
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Introduction |
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We have recently demonstrated that Ucn-I has beneficial short-term actions in experimental HF.8 Bolus administration of the peptide produced dose-dependent increases in cardiac output (CO) and reductions in peripheral resistance and arterial and ventricular filling pressures, in conjunction with improved renal function and decreased activation of vasoconstrictor peptides angiotensin-II, vasopressin, and endothelin-1. Repeated Ucn-I boli also induced repeated transient rises in the stress hormones, adrenocorticotrophic hormone (ACTH) and cortisol. The effects of long-term administration of Ucn-I in this setting need to be investigated before the therapeutic potential of the peptide can be realized. The present study examines for the first time the haemodynamic, hormonal, and renal effects of prolonged (days) administration of Ucn-I in experimental ovine HF.
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Methods |
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Study protocol
Each sheep received a continuous 4-day intravenous infusion of ovine Ucn-I (0.3 µg/kg/h) (American Peptide Company Inc., USA) and a vehicle control (0.9% saline) in a balanced, randomized, and crossover design (four animals to each sequence). Following induction of HF by rapid left ventricular pacing (225 b.p.m.) for 7 days,16 treatments were then administered over Days 812 of pacing. A week without pacing between phases allowed recovery to normal pre-pacing parameters. Infusions commenced at 1000 h on study Day 0 and were administered via the pulmonary artery catheter in a total volume of 50 mL/day.
MAP, LAP, CO, stroke volume (SV=CO/heart rate), and calculated total peripheral resistance (CTPR=MAP/CO) were recorded at 15 min intervals in the hour preceding infusion on study Day 0 (baseline), at 0.5, 1, 1.5, 2, 4, and 6 h following commencement of treatment, and then daily on study Days 14. Haemodynamic measurements were determined by on-line computer assisted analysis using established methods.17 Blood samples were drawn from the left atrium (immediately following haemodynamic measurements) into tubes on ice, centrifuged at 4oC and stored at 80oC before the assay for Ucn-I,8 cyclic adenosine monophosphate (cAMP), arginine vasopressin (AVP), ACTH, cortisol, atrial and brain natriuretic peptide (ANP and BNP, respectively), plasma renin activity (PRA), aldosterone, endothelin-1, and catecholamines.1618 All samples from individual animals were measured in the same assay to avoid inter-assay variability. Plasma electrolytes, glucose, and haematocrit were measured with every blood sample taken.
Urine volume and samples for the measurement of urine cAMP, sodium, potassium, and creatinine excretion were collected over the 2 h prior to infusion on study Day 0 (baseline), at 2, 4, and 6 h following commencement of treatment, and then daily on study Days 14. Water intake was measured as per urine output, and food intake was calculated daily.
The study protocol was approved by the local Animal Ethics Committee.
Statistics
A sample size of eight sheep was selected because previous studies of vasoactive hormones using a similar number of animals in this model of HF8,1618 consistently produce unequivocal results under these carefully controlled experimental conditions and consistently provide >80% power to detect >30% effects (differences between within-subject mean) at a two-tailed alpha level of 0.05.
Data are expressed as mean±SEM. Baseline haemodynamic and hormone values represent the mean of the four and two measurements, respectively, made within the hour immediately pre-infusion. Differences between non-paced laboratory normal sheep (n=20) and HF sheep (control baseline data) were compared using independent t-tests (Table 1), and differences between pre-treatment control and Ucn-I baseline data were analysed using paired t-tests. Temporal changes within the control data were investigated using repeated measures analysis of variance (ANOVA). Differences between control and Ucn-I treatments [acute (0.56 h) and chronic (14 days) effects tested separately] were analysed by ANOVA, where the period, sequence, treatment, and within-treatment times are all tested as fixed effects (treatment/time interactions quoted in text). The adequacy of the washout period between treatment phases was tested within this analysis by using the period, sequence and sequence by treatment terms. Possible within-animal correlations were taken into account by fitting a random intercept. Where significant differences were identified by ANOVA, the level of significance at individual time points on Figures 14 and Table 2 was determined by pair-wise least-significant difference (LSD) tests using the appropriate mean-square error term from the ANOVA (Fisher's protected LSD test). The sphericity assumption for the repeated measures analysis was confirmed using Mauchly's test. Statistical significance was assumed when two-tailed P<0.05.
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Results |
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Seven days of rapid left ventricular pacing induced the haemodynamic, hormonal, and sodium-retaining hallmarks of established HFwith reduced MAP, CO, SV, and renal function, increased CTPR and LAP, and ubiquitous hormone activation. A comparison of measurements made in sheep before (laboratory normal data, n=20) and after the development of pacing-induced HF (control baseline data) can be seen in Table 1.
A number of variables changed significantly over the 4 days of control treatment (with continued pacing) including decreases in CO (P<0.001), SV (P<0.001), MAP (P=0.0009), haematocrit (P<0.001), creatinine clearance (P<0.001), urine creatinine (P=0.0009) and urine potassium excretion (P<0.001), and in rises in LAP (P<0.001), norepinephrine (P=0.0006), aldosterone (P=0.0265), endothelin-1 (P=0.0201), BNP (P=0.0188), and plasma creatinine (P=0.033). In addition, on Day 1 of the control phase plasma cAMP (P=0.0057) and drinking (P=0.044) fell, and plasma ACTH (P=0.0014) and cortisol (P=0.039) rose acutely (Figures 14, Table 2). There was no statistical evidence of significant carry-over effects for any of the variables tested (P>0.05 for all sequence by treatment interactions), or of significant differences between pre-treatment control and Ucn-I baseline data.
When compared with vehicle control data, infusion of Ucn-I induced prompt (30 min) and sustained (4 day) increases in CO (Day 4: 1.8±0.2 vs. 2.3±0.2 L/min, P<0.001) and SV (7.8±0.8 vs. 10.2±1.0 mL/beat, P=0.0011), and reductions in MAP (72±3 vs. 70±3 mmHg, P=0.0305), LAP (26±1 vs. 11±2 mmHg, P<0.001), and CTPR (43±6 vs. 32±4 mmHg/L/min, P<0.001) (Figure 1). Haematocrit was elevated relative to control over Days 14 (P<0.001, Table 2).
Infusion of Ucn-I gradually increased circulating concentrations of the peptide from a baseline of 14 pmol/L to a plateau of 7500 pmol/L on Day 3 (P<0.001) (Figure 2). Plasma concentrations of Ucn-I's intracellular second messenger, cAMP, were unchanged during the first 24 h but were elevated when compared with control over Days 24 (Day 4: 30±2 vs. 35±2 nmol/L, P=0.0143). Ucn-I administration produced rapid but short-lived increases in plasma AVP (P=0.0129), ACTH (P=0.0057), and cortisol (P=0.048) on Day 0 (Figure 2), and while the latter two hormones remained close to control levels during Days 14, AVP concentrations were significantly reduced over this time compared with control (Day 4: 24±4 vs. 14±2 pmol/L, P=0.0028).
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Ucn-I infusion acutely increased urine output (46 h: 2-fold, P=0.0254), urinary potassium excretion (1.8-fold, P=0.001), sodium excretion (5.5-fold, P=0.0007), creatinine excretion (1.4-fold, P=0.0046), and creatinine clearance (1.3-fold, P=0.0031) (Figure 4 and Table 2), with the latter three variables remaining elevated compared with control over the following 4 days of treatment (Day 4: sodium excretion 5.3-fold, P=0.0001; creatinine excretion 1.2-fold, P=0.005; creatinine clearance 1.3-fold, P=0.0055). Urine output and urine cAMP excretion tended to be raised over this period (P=0.0748 and P=0.0619, respectively). Water intake was reduced short-term (P=0.0491) but was not significantly lower than control over the remaining 4 days, whereas food intake was attenuated over the first 2 days of Ucn-I infusion (P=0.0283), before returning to pre-treatment levels over Days 3 and 4 (Figure 4).
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Discussion |
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The haemodynamic effects of protracted infusion of Ucn-I in sheep with HF are qualitatively similar to those we have reported formerly in response to bolus administration of the peptide in our ovine HF model.8 The moderate yet persistent blood pressure-lowering effects of infused Ucn-I in the present study occurred in association with substantial reductions in CTPR, indicating an effect on arterial tone. This is in agreement with in vitro studies demonstrating a direct vasodilator action of the peptide in both rat and human vasculature.3,19 The persistence of this hypotensive response (4 days) concurs with reports in normal mice where pre-treatment with Ucn-I for 3 days did not diminish the blood-pressure lowering effect of subsequent challenges of the peptide.20 Other mechanisms possibly contributing to the prolonged hypotensive action of Ucn-I in these HF sheep include plasma volume contraction, as evidenced by the rise in haematocrit, as well as marked suppression of a number of vasoconstrictor factors (activated in this setting) including endothelin-1, AVP, and reninangiotensinaldosterone.
Ucn-I-induced increases in CO were prompt and also sustained for the duration of the 4-day study period. Although this response may be due to the reduction in peripheral resistance and MAP, it is likely other actions of Ucn-I, including positive inotropism (as reported previously in the isolated rat heart12 and evidenced by the significant rise in SV in the present study) play an important role. Ucn-I also exhibits chronotropic effects, which we have described in earlier studies in normal sheep8 but are unable to demonstrate in these animals with pacing-induced HF as the heart rate is fixed at 225 b.p.m. to maintain a stable HF profile.21 Although it is possible that the combination of Ucn-I's chronotropic and inotropic effects may have an adverse impact on myocardial oxygen demand (and thus may in part counterbalance the peptide's other benefits in HF), additional actions of Ucn-I to dilate coronary arteries,12 improve cardiac bioenergetics (preservation of high-energy phosphate stores)15 and lessen cardiac workload through the reduction of peripheral resistance and blood pressure, may offset this unfavourable consequence. Furthermore, it is likely that Ucn-I treatment would be used in conjunction with beta-blocker therapy. The cardiac and vascular actions of Ucn-I appear to be mediated largely via activation of local CRF-R2 receptors and production of adenylate cyclase12,19with both activities prevented by CRF-R2 antagonism12 and absent in CRF-R2 knockout mice.22 Of note, CRF-R2-deficient animals exhibit hypertension when compared with their wild-type littermates.23
Four-day infusion of Ucn-I also resulted in an impressive halving of LAP, presumably a reflection of the substantial increase in CO, concurrent haemoconcentration (due at least in part to a reduction in water intake and rise in urine output), and likely lusitropic actions of Ucn-I (as shown for Ucn-II).24 A possible contribution from reduced venous tone cannot be excluded from our data. Indeed, recent reports suggest that Ucn-I may be of significance in the regulation of the venous circulation in humans.11 However, in the absence of direct left ventricular imaging in this experiment, a significant drop in left ventricular enddiastolic volume is conjectural. Our results point towards a role for Ucn-I in both the short- and long-term regulations of haemodynamic function, and demonstrate that extended augmentation of this peptide produces a sustained and desirable haemodynamic profile in experimental HF.
Prolonged elevation of circulating Ucn-I was associated with significant rises in plasma levels of cAMPa proposed intracellular second messenger of the peptide.19 Plasma cAMP is elevated in HF in relation to the severity of the disease,25 and concentrations fall following successful HF treatment (with agents which do not utilize the cAMP pathway).25 The fact that we observe haemodynamic improvement in conjunction with increased plasma cAMP following Ucn-I treatment in the present study, rather than a decrease in nucleotide levels, suggests that the rise in cAMP is (directly) attributable to Ucn-I. There was, however, a temporal dissociation between the onset of haemodynamic and hormonal effects (1530 min) and plasma cAMP increases (2 days), a finding we have observed previously following short-term administration of the peptide,8 suggesting that cAMP was raised sufficiently at the tissue level to induce the significant responses that ensued. Alternatively, other signalling pathways may be involved.10,12
Stimulation of the hypothalamic-pituitary-adrenal (HPA) stress axis via activation of CRF-R1 is a well-documented action of Ucn-I, yet obviously undesirable when considering the peptide's potential as a therapeutic strategy in cardiovascular disease. Although infused Ucn-I did induce a significant rise in plasma ACTH and cortisol in the present investigation (the former presumably via direct stimulation within the pituitary and potentiation by concomitant increases in AVP),26 the rises were transient (12 h) and both hormones persisted at pre-infusion levels for the following 4 days. This response differs from that observed following intermittent Ucn-I administration in the ovine HF8 where each bolus resulted in an acute marked rise in ACTH/cortisol. It is likely that deletion of the pituitary pool of readily available ACTH and negative feedback by cortisol, followed by receptor down-regulation and desensitization,27 diminished the ACTH response to further stimulation by Ucn-I. The plasma AVP response appeared to be biphasicwith an acute rise (0.52 h) followed by a considerable reduction (from elevated baseline concentrations) over Days 14 of the infusion. Significant attenuation of plasma AVP has previously been observed following bolus administration of the Ucn-I in HF sheep,8 and is likely mediated via improved CO and pressure at sinoaortic volume receptors, and possibly also through reductions in plasma angiotensin-II (as reflected by declining PRA).28
Similar to acute administration of Ucn-I,8 we observed marked reductions in the vasoconstrictor reninangiotensinaldosterone and endothelin-1 systems with long-term Ucn-I treatment. PRA decreases were sustained over the 4-day infusion period and occurred in the face of falls in arterial pressure (and plasma concentrations of AVP and the natriuretic peptides). Whether PRA reductions were due to increased delivery of sodium to the macula densa (evidenced by the significant rise in sodium excretion), a direct inhibitory effect of the Ucn-I on renin secretion, or some other PRA-inhibitory mechanism remains to be determined. Although attenuation of the plasma aldosterone is likely to be in part a consequence of the declines in circulating PRA/angiotensin-II, it is important to note that the fall preceded that of PRA, suggesting a possible direct inhibitory effect of Ucn-I on aldosterone secretion. The mechanism behind the striking and persistent reductions in plasma endothelin-1 concentrations we observed cannot be established from our data. Although it has been shown that Ucn-I opposes the vasoconstricting actions of endothelin-1,10,11 the peptide's effect on endothelin secretion is unknown. Clearly, further investigation into the relationships between Ucn-I and these clinically important vasoactive systems is required.
Despite significant acute falls in LAP (and therefore reduced cardiac transmural pressure and stimulus for secretion), plasma natriuretic peptide levels were unchanged during the first 6 h of the Ucn-I infusionfindings in contrast with previous studies showing close parallelism between falls in atrial pressure and circulating ANP/BNP concentrations following the administration of vasodilator agents.29 It is conceivable that Ucn-I enhanced natriuretic peptide secretion2 sufficiently during this time to counteract the opposing effect of falling intracardiac pressure, whereas the significant ANP/BNP reductions over Days 14 reflect the substantial and sustained improvements in haemodynamic function. It should also be noted that plasma ANP/BNP tended to rise slowly again during Days 24, over which time LAP was observed to be fairly constant.
The renal response to extended Ucn-I treatment in sheep with HFa sustained natriuresis, increase in creatinine clearance and a trend for augmented urine outputis similar in type to those observed following acute administration of the peptide,8 and occurred despite falls in arterial pressure (and therefore renal perfusion pressure) and reductions in plasma ANP/BNP. It is likely that the prominent and persistent reductions in circulating levels of the sodium/volume-retaining factors AVP, angiotensin-II, and aldosterone (and perhaps endothelin-1) contributed to these renal responses. Ucn-I's effect on renal haemodynamics, a protracted elevation in the glomerular filtration rate (as assessed by the increase in creatinine clearance) and possibly renal blood flow,10 may also have contributed to the sustained natriuresis (and diuresis). In addition, direct tubular actions may have been involved, given reports of Ucn-I expression within the kidney30 and the relative increase in urine cAMP excretion observed in the present study. The maintenance of sodium excretion in conjunction with the extended decrease in renal perfusion pressure points to a shift in the pressure-natriuresis curve with long-term Ucn-I treatment in HF. These renal effects are clearly of benefit in this underperfused, sodium/volume-retaining state.
Four-day Ucn-I also suppressed food and water intake in these sheep with HF. Although appetite suppression may be considered an adverse side effect of any potential therapeutic agent in HF, we found that inhibition of feeding was transient, with the peak effect apparent over Days 1 and 2. These data are in agreement with studies investigating more prolonged Ucn-I administration in the normal mice31,32 and suggest that tolerance to Ucn-I-induced appetite suppression occurs. Indeed, Cohen et al.20 reported that repeated administration of Ucn-I resulted in a reduced effect to inhibit food consumption, whereas the hypotensive action of the peptide persisted. Consistent with these findings, transgenic mice lacking the CRF-R2 receptor exhibit elevated basal blood pressure,23 but normal basal feeding and weight gain,22 suggesting that the hypotensive and appetite-suppressant effects of Ucn-I are mediated by different mechanisms. Similar to the findings with acute Ucn-I administration,8,33 we also observed a tendency for plasma glucose levels to be elevated when compared with the control during extended Ucn-I treatment, with the peak effect (Days 1 and 2) matching that of food suppression. The underlying mechanisms by which Ucn-I decreases food intake are unknown but may involve reduced gastric emptying31 as well as interaction with other factors known to regulate appetite, such as leptin34 and ghrelin.35 The repressed drinking response we noted in the present study was also transitory, an observation made previously in mice following 14-day Ucn-I treatment,32 and thought to be secondary to food intake reductions.36
The progressive haemodynamic and renal deterioration and augmented activation of many of the hormones systems noted during the control phase of the present study presumably reflect the increasing severity of HF resulting from continued rapid cardiac pacing. The acute rises in plasma ACTH and cortisol (on Day 1 of the control phase) are likely a consequence of the mild pyrogenic reaction that occurs in some of these chronically instrumented animals following flushing of the fluid-filled lines,37 while the fall in cAMP levels may relate to diurnal variation.38
In addition to the beneficial haemodynamic, hormonal and renal effects demonstrated in the present study, Ucn-I appears to have a direct cardioprotective role. As mentioned earlier, Ucn-I reduces myocyte cell death caused by ischaemia/reperfusion in vitro, ex vivo, and in vivo.14,15 Ucn-I-induced cardioprotection appears to involve several mechanisms including enhanced cardiac expression and production of another cytoprotective peptide, cardiotrophin-1,39 which is also raised in the circulation of the patients with heart disease.40 The protective effects of both peptides involve activation of the p42/p44 MAPK and PI-3 kinase/Akt pathways. Ucn-I cardioprotection may also include stimulation of heat shock protein 9041 and the natriuretic peptides,2 and attenuation of calcium-insensitive phospholipase A2 gene expression.42
Although the cardioprotective actions of Ucn-I undoubtedly enhance its potential as a treatment for HF, reports that the peptide also possesses cardiac hypertrophic activity43 are counter intuitive to Ucn-I's use in this disease. However, a recent report by Davidson et al.43 demonstrating that Ucn-I induces hypertrophy in cultured rat cardiac myocytes via a pathway distinct from that which mediates its protective actions (following exposure to transient ischaemia) suggests it might be possible to separate these two effects, and thus enhances the survival of cardiomyocytes in hypoxic conditions while avoiding undesirable activation of the hypertrophic pathway. Interestingly, Ucn-I is also reported to attenuate the proliferation of vascular smooth muscle cells.44 Clearly, continued investigation into Ucn-I's cellular effects is essential.
In 2001, another peptide structurally related to Ucn-I was identified, termed Ucn-II.45 Much interest is currently being directed towards this latter peptide as a potential therapeutic agent in HF due to its specificity for CRF-R2,45 and therefore minimal undesirable activation of the CRFR1-mediated HPA axis. However, until the mechanisms (and receptor sub-types) underlying the salutary attenuation of vasoconstrictor/volume-retaining hormone systems and augmentation of renal function seen following Ucn1 treatment in the present study are identified, continued investigation of this peptide is crucial. In addition, the transient nature of the ACTH/cortisol rise induced by sustained Ucn-I administration may suggest that treatment with the more selective CRF-R2 ligand, Ucn-II, may not be all that more advantageous than Ucn-I as a therapy for congestive HF.
In conclusion, we have demonstrated for the first time that extended augmentation of the Ucn-I system in experimental HF induces sustained and beneficial reductions in cardiac preload and MAP, improvements in CO and renal function, and inhibition of a range of adverse vasoconstrictor/volume-retaining factors. In contrast, less desirable stimulation of the HPA axis and appetite suppression were transient events. These findings point towards a role for Ucn-I in long-term pressure/volume regulation and, in combination with its cardioprotective activity, suggest that the peptide has therapeutic potential in HF.
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Acknowledgements |
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