Cardiovascular disease and its complications, including heart failure (HF), remain a leading cause of death and disability in the western world. With our ageing population, the incidence and prevalence of HF are on the rise, as is its attendant mortality and morbidity, making the exploration and development of new therapeutic interventions imperative. Neurohormonal activation is a characteristic feature of HF, and advances to date in the prevention and treatment of this disease have centred on the manipulation of a number of hormone systems 1.
Animal models have been and will continue to be crucial to our understanding of the pathophysiology of cardiac disease and in the development of therapeutic strategies. Numerous experimental models of HF exist, with a range of species and interventions used to mimic the disease. The rapid ventricular pacing model of HF in sheep 2, on the basis of the pioneering studies of Whipple et al. 3 in dogs, produces the haemodynamic, hormonal and metabolic hallmarks of acute and severe low-cardiac output HF in humans. The progression of HF indicates time, heart rate and species dependency. In sheep, sustained left ventricular pacing at 225 beats/min for 7 days produces a marked decrease in cardiac output in association with reductions in arterial pressure, elevated systemic peripheral resistance and cardiac preload, widespread neurohumoral activation, avid sodium and water retention, decreases in glomerular filtration rate and development of pulmonary congestion and oedema. This model in an intact, conscious large animal is one of the few that provides a faithful replication of many aspects of severe human HF. It is unique in allowing both the development and the resolution of the condition to be studied in the same animal with serial measurement of key haemodynamic, hormonal and renal variables. It also enables investigations in varying states of cardiac dysfunction – easily altered by titration of the pacing rate. Importantly, the large body size and blood volume of the sheep, compared with many other experimental animals, facilitates haemodynamic and urine measurements and allows repeated blood sampling for the measurement of a wide range of hormones, all of which are essential to adequately determine inter-relationships. In addition, sheep are docile and adjust well to handling, and yet are robust with respect to surgery and infection (http://www.adelaide.edu.au/ANZCCART/publications/A9_SheepFactSheet.pdf).
Over the past 25 years, the Christchurch Heart Institute has utilized the ovine model extensively in investigations into the actions and regulation of key and novel hormones involved in the control of blood pressure and fluid volume in both cardiovascular health and disease, with a focus on ways of manipulating these hormones in order to prevent and treat HF. A variety of hormones have been studied to date, including the natriuretic peptides, the renin–angiotensin–aldosterone system, endothelin and adrenomedullin peptides. More recently, attention has expanded to encompass the urocortin (Ucn) family – a group of peptides whose role in normal cardiovascular physiology and disease states is now increasingly being recognized. In this review, we focus on our research into the Ucns to show the range of preclinical studies carried out in sheep exploring the cardiovascular physiological and pathophysiological significance of hormones and their therapeutic potential in HF.
Ucns 1, 2 and 3 are a group of endogenously produced peptide hormones belonging to the corticotrophin-releasing factor (CRF) family. The peptides are highly preserved across species, and comparison between the human and ovine forms of Ucn1, Ucn2 and Ucn3 shows 95, 76 and 89% homology, respectively (Table 1). The Ucns exert their effects through CRF G-protein-coupled receptors type 1 (CRF-R1) and type 2 (CRF-R2), with the Ucn2 and Ucn3 paralogues reported to bind selectively to the CRF-R2 subtype that mediates cardiovascular actions. Both the peptides and the CRF-R2 receptor are distributed widely in tissues and organs involved in pressure/volume regulation, most notably the heart, vasculature, kidneys, adrenals and gastrointestinal tract. In normal animals, they have been shown to cause relaxation of the vasculature and exert positive inotropic and lusitropic effects on the heart. This makes the Ucns a potentially attractive target in the treatment of HF. In addition, Ucn1 gene expression and protein are increased in the failing human heart in inverse relation to cardiac function, suggesting not only that the peptides may play a role in the pathophysiology of HF but that they may also have prognostic significance 4,5.
Plasma levels: sheep to man
Elevated plasma concentrations of Ucn1 in the setting of chronic HF were first reported in 2002 in the ovine model using a locally developed radioimmunoassay, with levels measuring 12±1 pmol/l in normal animals versus 20±2 pmol/l in HF 6. This finding provided further evidence indicating a pathophysiological involvement of the peptides in the disease. Later investigations into the source of circulating Ucn1 through transorgan arteriovenous sampling both before and after the development of pacing-induced HF showed a net production of the peptide across the hepatic, renal and hind limb tissue beds in both states – with proportional step-ups greater in HF, and a significant increase across the head also observed in HF (with a similar trend across the heart) 7. These data suggested the widespread ‘constitutive’ synthesis and release of Ucn1 consistent with the peptide’s ubiquitous tissue distribution.
Subsequent studies in man by our laboratory reported comparable plasma levels and increases (to those noted in sheep) in healthy human volunteers (∼7 pmol/l) and symptomatic HF patients (∼14 pmol/l) using a similar assay configuration 8. In this study, not only were Ucn1 levels an independent predictor of a final diagnosis of HF in patients presenting to primary care with recent-onset dyspnoea or peripheral oedema, but concentrations were increasingly elevated with worsening New York Heart Association (NYHA) functional class (measuring 11.5±3.5, 12.4±2.5, 14.3±4.3 and 14.6±5.0 pmol/l in NYHA I through IV). In addition, plasma Ucn1 correlated negatively with left ventricular ejection fraction and positively with other circulating markers of HF severity including B-type natriuretic peptide (BNP). Further work showed that high plasma concentrations of the peptide are associated with poorer long-term clinical outcomes in patients with chronic systolic HF 9 and with increased mortality following acute myocardial infarction 10. Moreover, when combined with plasma NT-proBNP, Ucn1 enhanced risk stratification for the diagnosis of HF 9 and prognostic performance after acute myocardial infarction 10. These findings indicate a potential role for Ucn1 as a complementary biomarker for diagnosis and/or prognosis in cardiovascular disease.
In a regional blood sampling study carried out in 25 patients undergoing clinically indicated coronary angiography (using the assay referred to above), findings largely similar to those observed in sheep were noted – with net production of Ucn1 detected from heart, kidney, liver, head and neck and leg 5.
Administration studies: sheep to humans
Preclinical investigations into the integrated biological effects of all three Ucns have been performed in the ovine model. In a series of studies in sheep with severe pacing-induced HF, intravenous bolus doses of 10, 50 and 100 μg of Ucns 1, 2 (Fig. 1) and 3 all dose-dependently augmented cardiac output and reduced systemic vascular resistance, arterial pressure and cardiac filling pressures 6,12,13. These favourable haemodynamic effects were accompanied by suppression of a broad spectrum of markedly activated circulating vasoconstrictor/volume-retaining factors (including renin, angiotensin II, aldosterone, endothelin-1 and vasopressin) and improvement in renal function (including increases in urine volume, sodium excretion and glomerular filtration). Of note, identical doses of the peptides in normal animals elicited more limited bioactivity – with modest haemodynamic responses and negligible effects on vasoconstrictor hormones and renal parameters 6,12,13. The results of these studies supported a role for the Ucns in pressure/volume homoeostasis in HF and pointed towards a potential therapeutic application of this peptide in clinical cardiac failure.
The beneficial profile of effects found with acute Ucn1 and Ucn2 dosing was sustained over 4 days of treatment in severe HF 14,15, and occurred in conjunction with reduced ventricular gene expression of factors/pathways involved in cardiac hypertrophy (Gata 4; β-myosin heavy chain) and fibrosis (transforming growth factor-β; collagen 1), suggesting that the peptide exerts cardioprotective effects also at the molecular level 16. A role for endogenous Ucns as a protective compensatory response in HF was further bolstered by the exacerbation of deleterious haemodynamic and hormonal alterations observed upon blockade of CRF-R2 17, whereas administration of Ucn1 from the outset of rapid pacing appreciably delayed the development of overt HF in this model 18.
Comparison of the pharmacodynamics and pharmacokinetics of the three Ucns indicates that although the range (and to some degree, magnitude) of biological responses is largely comparable in sheep, the onset (Ucn3>Ucn2>Ucn1) and duration (Ucn1>Ucn2>Ucn3) of action differ appreciably. This is consistent with an extended biological half-life for Ucn1 relative to Ucn2 and Ucn3, in association with a lower clearance rate. However, steady-state volumes of distribution were found to be similar for Ucn1 and Ucn2, but considerably larger for Ucn3 19. Importantly, generally lower EC50 estimates were shown for Ucn2 and Ucn3, suggesting that these peptides are possibly more potent than Ucn1. These data provide valuable comparative information that may aid in the rational design of future clinical studies.
Work in sheep spearheaded several studies investigating the effects of the Ucns in humans. Whereas Wiley and Davenport 20 reported that all three peptides were essentially equipotent in relaxing isolated, endothelium-denuded human artery segments in vitro (although Ucn2 and Ucn3 produced the greatest maximum responses), the peptides do not appear to show similar potencies when administered in vivo. Short-term administration of Ucn1 at doses of 50 μg produced no significant haemodynamic or renal effects in either normal participants or patients with stable HF 21,22, although modest increments in plasma corticotropin and cortisol were observed in both states (mediated through CRF-R1). In contrast, Ucn2 administered at both 25 and 100 μg to humans with and without HF induced clear dose-related increases in cardiac output and left ventricular ejection fraction in conjunction with sizeable decreases in systemic vascular resistance (Fig. 1) and mean arterial pressure (∼19 mm Hg reductions in the HF setting) 11,23. A decrease in left ventricular dimensions and calculated cardiac work was also observed in the HF group. In contrast to findings in HF sheep, Ucn2-induced neurohormonal and renal effects in these patients were negligible 23. This disparity in responses likely reflects the relatively mild, stable and treated state of the patients recruited in these Ucn studies compared with the more extreme dysfunction (and otherwise nonmedicated condition) present in the ovine HF model. However, the lack of an increase in renin or catecholamine and the maintenance of renal indices, in the face of pronounced Ucn2-induced decrease in blood pressure (and thus renal perfusion pressure), does suggest a relative suppression of the renin–angiotensin–aldosterone and catecholamine systems as well as an augmentation of kidney function in these patients. In a very recently completed placebo-controlled pilot study in 53 patients admitted within 36 h for acute decompensated HF, 4 h Ucn2 infusions (5 ng/kg/min) markedly reduced peripheral vascular resistance (without compensatory tachycardia), improved cardiac output and inhibited aldosterone. In addition, Ucn2 induced a delayed diuresis and natriuresis and enhanced responses to super-added loop diuretic treatment 24,25.
With respect to Ucn3, to date, there is a single in-vivo study in humans that compared the vasodilator effects of Ucn3 and Ucn2 in the forearm arteries of healthy human volunteers, and found that although both peptides produced dose-dependent arterial vasodilatation, a 300-fold-higher dose of Ucn3 was required to induce a response comparable with that induced by Ucn2 26. Despite obvious differences in the potencies of the Ucn peptides in sheep and humans (presumably because of species-specific differences in peptide and receptor structures and affinities 27), there are still pharmacodynamic and pharmacokinetic similarities evident between the species – with the onset and duration of (vasodilator) response to Ucn3 more rapid and less prolonged relative to Ucn2 in humans 26, and Ucn1 showing a longer half-life, greater volume of distribution and reduced clearance rate compared with Ucn2 11,21–23.
With the clinical focus directed towards Ucn2, and as any prospective new therapy in HF will likely be used in addition to current established treatments, the combination of the peptide with angiotensin-converting enzyme inhibitors, diuretics and blockers of β-adrenergic and mineralocorticoid receptors have all been assessed in the sheep model. In every case, the combination resulted in enhancement in salutary haemodynamic effects compared with either agent alone, together with amelioration of the undesirable effects of existing therapies and without causing any major adverse effects (such a marked decrease in arterial pressure, which is a frequent and unwanted threat when combining multiple agents in HF). The combination of Ucn2 with an angiotensin-converting enzyme inhibitor attenuated increases in plasma renin, further reduced aldosterone, enhanced increases in cardiac output and induced additional decreases in systemic resistance 28. Ucn2 together with the loop diuretic furosemide augmented renal responses without exacerbation of potassium loss and with reduced renin activation 29. Ucn2 added to β-blockade prevented the initial decreases in cardiac output and renal function consequent upon introducing β-blockers in severe HF, in association with augmented suppression of aldosterone 30. Ucn2 combined with mineralocorticoid receptor blockade reduced plasma renin activity, angiotensin II and aldosterone levels, improved renal function and prevented plasma potassium increases 31.
Additional work in conscious sheep has examined the impact of the Ucn peptides on sympathetic traffic directed towards the heart by recording electrodes implanted in the cardiac sympathetic nerves. Intravenous administration of all three Ucns was found to significantly reduce cardiac sympathetic nerve activity and, in the case of Ucn2, this was apparent at doses below the threshold for inducing changes in blood pressure and heart rate 32–34. These findings raise the possibility of a specific therapeutic benefit from the Ucns in states such as cardiac injury (as well as HF) in which sympathetic overdrive is both proarrhythmic and contributes towards ventricular remodelling, progressive cardiac dysfunction and increased mortality.
In summary, the sheep, with its large body size and blood volume, provides us with an excellent model with which to investigate hormones implicated in the pathophysiology of HF. A range of studies have been carried out using this ovine model, including investigations into the function of the endogenous peptide (through a blockade of its activity), measurement of circulating levels and bioactivity (with a focus on integrated haemodynamic, endocrine and renal effects and inter-relationships) in both cardiovascular health and disease, pharmacodynamic and pharmacokinetic profiles and mechanisms of action – all of which help establish the pathophysiological role and therapeutic potential of a hormone in HF.
Conflicts of interest
There are no conflicts of interest.
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