Aldosterone - Cardio Symposium 2009 · Aldosterone (Aldo) is the main regulator of sodium...

R E P O R T

1st Human and Veterinary Crossover Symposium

on Aldosterone

December 2009

cardiologycardiology

AT THE HEART OF INNOVATION

FirstHuman and VeterinaryCrossover Symposium

on Aldosterone

The contribution of aldosterone to heart failure was the focus of a recent symposium, hosted by Ceva Santé Animale. The principle of this symposium was to promote expertise exchanges between human and veterinary medicine. The programme included eminent cardiology researchers from around the world, both from the human and veterinary fields. You will find in this report a summary of the presentations and the discussions that took place, mixing state of the art science with the most recent clinical developments in man and animals.

We hope that you will both enjoy and benefit from the research compiled herein.

Sylvie Bourrelier and Emilie GuillotCorporate Marketing – Veterinary Technical Support Ceva Santé Animale

Speakers and chairs at the meeting, from left to right: Frédéric Jaisser,Nicolette Farman, Faiez Zannad, Bertram Pitt, Jonathan Elliott, Mark Oyama, Johann Bauersachs, Claudio Bussadori, John Bland and Robert Hamlin

p4 Aldosterone and mineralocorticoid receptors: expanding views from the kidney to the cardiovascular system

Nicolette Farman

p8 Overexpression of the mineralocorticoid receptors: pathophysiological consequences

Frédéric Jaisser

p16 Mineralocorticoid receptor blockade: how experimental evidence supports the clinical benefit

Johann Bauersachs

p22 The proximate cause of heart failure: models, remodeling, and re-remodeling

Robert L. Hamlin

p28 After RALES, the journey continues

Bertram Pitt

p34 Efficacy of spironolactone in dogs with naturally occurring Myxomatous Mitral Valve Disease

Claudio Bussadori

p40 Principles of survival analysis illustrated by CEVA’s spironolactone trials

John Martin Bland

p52 Current clinical status and future directions for biomarkers in heart failure

Faiez Zannad

CONTENT / ABSTRACTS & QUESTION SESSIONS

4 - 1st Human and Veterinary Crossover Symposium on Aldosterone 1st Human and Veterinary Crossover Symposium on Aldosterone - 5

Nicolette Farman is Research Director (Senior Investigator) at INSERM (French National Institute of Health and Medical Research). She joined the public research body in 1978 (INSERM U2, Limeil Brévannes Hospital) and was a Board Member between 2001 and 2008.

She is a Member of the French Society of Nephrology, American Society of Nephrology, American Physiological Society and Honorary Member of the South-American Society of Nephrology. She is referee for several peer-review journals such as J. Am Soc. Nephrology, Am. J. Kidney Diseases, Endocrinology, Nature.

Nicolette Farman has organized four international congresses deal ing with aldosterone in France and abroad. She was one of the pioneers in discovering the cardio-vascular effects of this hormone. She received the title of “Knight of National Order of Merit” and the Award of the Academy of Medicine in 2001. She is currently working alongside Dr Frédéric Jaisser at Unit 872 of INSERM.

Aldosterone And minerAlocorticoid receptors:eXpAndinG VieWs From tHe KidneY totHe cArdioVAscUlAr sYstem

The steroid hormone aldosterone plays a major role

in the control of blood pressure and extracellular

volume homeostasis 1. Aldosterone is synthesized in

the glomerular zone of the adrenal cortex in response

to hyperkaliemia or sodium depletion, as the endpoint

of activation of the renin-angiotensin system.

Considerable work has been achieved to improve the

understanding of the molecular and cellular events

involved in renal aldosterone actions, in normal as

well as in pathological situations 2,3.

It is well established that aldosterone stimulates

renal sodium reabsorption and potassium excretion,

an effect that can be blocked by administration of

the mineralocorticoid receptor (MR) antagonist

spironolactone. These effects are observed 1-2hrs

after hormone administration, a delay required

for hormonally-mediated activation of sodium

transporters and regulation of transcription of

target genes. Aldosterone binds to the MR, which

is a ligand-dependent transcription factor that

belongs to the nuclear receptor superfamily. Once

translocated into the nucleus, hormone-receptor

complexes bind to glucocorticoid response elements

within the promotor region of early aldosterone-

induced genes to modulate their transcription. This

triggers an increase in the activity and number of

sodium transporters or channels. In a typical target

cell for aldosterone of the distal nephron, such as

the renal collecting duct principal cell, the sodium of

the tubular fl uid enters the cell through amiloride-

sensitive apical sodium channels, and it then

extruded from the cell to the peritubular space by the

Na-KATPase located in the basolateral membrane.

MR antagonists (spironolactone, eplerenone)

prevent aldosterone binding and thus are useful

therapeutic tools to prevent inappropriate MR

activation. The MR has been cloned in 1987 4, and

it was evidenced that the MR displays similar high

affi nity (in the nanomolar range) for aldosterone

and glucocorticoid hormones, that are much more

abundant in the plasma (100-1000 fold) than

aldosterone. This should lead to permanent illicit

occupancy of the MR by glucocorticoid hormones,

inducing permanent maximal sodium retention,

independent of plasma aldosterone levels. The

MR protector enzyme 11 ß hydroxysteroid

dehydrogenase type 2 (11-HSD2, coexpressed with

the MR in cells of the distal nephron) metabolizes

circulating glucocorticoid hormones (cortisol in man,

corticosterone in rodents) into inactive 11-dehydro

derivatives (cortisone, 11-dehydrocorticosterone)

with very low affi nity for the MR.

More recently it became apparent that the MR is also

expressed in the cardio-vascular system, together

with low HSD2 5 and the nature of aldosterone

action in these tissues is actively investigated,

in normal as well as in pathological situations.

nicolette FarmanMD, PhD

Research Director (Senior Investigator) – INSERM (French National Institute of

Health and Medical Research).

Paris, France

Contact: [email protected]


Beside the cardiovascular consequences of an

altered functioning of the renin-angiotensin system

leading to hypertension, direct cardiovascular effects

of aldosterone have been evidenced 6. There are

many data in favour of an involvement of aldosterone

in cardiac remodelling and fibrosis 7. Exposure of

uninephrectomized rats to aldosterone and high salt

diet leads to cardiac fibrosis that can be prevented

by spironolactone. Importantly, the RALES and

the EPHESUS studies 8,9 have demonstrated the

beneficial effect of MR-antagonists (spironolactone,

eplerenone) in large cohorts of patients with severe

heart failure or following myocardial infarction.

These initial observations have now opened a major

new field to improve treatments of cardiac failure in

patients. Major efforts are now made to elucidate

aldosterone impacts on cardiovascular functions.

References

1. Farman N, Rafestin-Oblin ME. Multiple aspects of mineralocorticoid selectivity. Am J Physiol. 2001;280:F181-192.

2. Connell JM, Davies E. The new biology of aldosterone. J Endocrinol. 2005;186:1-20.

3. Pascual-Le Tallec L, Lombes M. The mineralocorticoid receptor: a journey exploring its diversity and specifi city of action.

Mol Endocrinol. 2005;19:2211-2221.

4. Arriza JL et al. Cloning of human mineralocorticoid receptor complementary DNA: structural and functional kinship with the glucocorticoid receptor.

Science. 1987;237:268-275.

5. Lombes M et al. Prerequisite for cardiac aldosterone action. Mineralocorticoid receptor and 11ß-hydroxysteroid dehydrogenase in the human heart.

Circulation. 1995; 92:175-182.

6. Brilla CG, Weber KT. Mineralocorticoid excess, dietary sodium, and myocardial fi brosis. J Lab Clin Med. 1992;120: 893–901.

7. Funder JW. Aldosterone, mineralocorticoid receptors and vascular infl ammation. Mol Cell Endocrinol. 2004; 217: 263–269.

8. Pitt B et al. 1999 The effect of spironolactone on morbidity and mortality in patients with severe heart failure. N Engl J Med. 341: 709–716.

9. Pitt B et al. 2003 : Eplerenone, a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction.

N Engl J Med. 348: 1309–1321.

Aldosterone And minerAlocorticoid receptors: eXpAndinG VieWs From tHe KidneY to tHe cArdioVAscUlAr sYstem


Frédéric Jaisser is Research Director at the Unit 872 of INSERM (Institut National de la santé et de la Recherche Médicale), the French public research body entirely dedicated to human health. He is Professor at the Faculty of Medicine of Reims where he coordinates different courses such as « Animal Models and Pathophysiological Mechanisms ».

He is MD, specialist in Nephrology and has a University degree in Biological and Medical Engineering. He joined INSERM in 1996. His fields of expertise are mainly renal and cardiovascular pathophysiology, development of transgenic animal models for pathophysiologic studies or human disease models. He is also the coordinator of several multicentric projects.

He i s an Ed i tor i a l Board Member o f Endocrinology and an expert for several other peer-review journals such as Circulation or Hypertension. He is currently the President of ESAC-France (European Section of the Aldosterone Council).

oVereXpression oF tHe minerAlocorticoid receptor:pAtHopHYsioloGicAl conseqUences

1- Mineralocorticoids and Cardiovascular diseases

Aldosterone (Aldo) is the main regulator of sodium

reabsorption 1. The mineralocorticoid receptor (MR)

is activated by Aldo binding and is targeted to the

nucleus where it binds to Glucocorticoid (Gluco)

Responsive Elements and triggers gene transcription.

New targets of Aldo have been discovered in

the last decade, that extends its actions to the

cardiovascular (CV) system 2. We have shown that

MR is expressed in cardiomyocytes, coronary vessels,

aorta and resistance arteries. MR is expressed in

both endothelial and smooth muscle cells where

its functions are still poorly understood 3. Vascular

MR activation, as addressed in pharmacological

experimental models or in human volunteers and

patients with CV diseases, has been proposed to

be linked to endothelial dysfunction and vascular wall

remodeling 4, 5.

The RALES and EPHESUS clinical trials have

demonstrated that the addition of MR antagonists

to standard care markedly reduce the overall and

cardiovascular mortality in patients with heart failure

or in acute myocardial infarction complicated by left

ventricular dysfunction, respectively 6, 7. Moreover, MR

antagonism has been recently shown to be beneficial

in diabetic nephropathy, as well as in chronic renal

diseases 8. The beneficial effects of MR antagonists

in CV diseases are explained mainly by a decrease

of cardiac fibrosis and an improvement of peripheral

vascular function. However, direct effect on cardiac

myocytes, endothelium and smooth muscle cells are

still underscored.

Plasma Aldo levels are not correlated with the clinical

response to MR antagonists. This may be explained by

an increase in MR expression/MR activation in heart,

vessel and kidney as reported in human diseases

and experimental animal models. MR expression is

increased in heart after myocardial infarction, heart

failure and hypertension 9, 10. In the vessels MR

expression is increased in arterioles of SHR rats 11.

MR activation (in absence of increased Aldo or MR

expression) has also been proposed to be related to

cellular oxidative stress.

The signaling pathways involved in mineralocorticoid

effects in CV diseases remain elusive because: 1) most

of the proposed signaling pathways have been identified

in cultured cells; 2) their identification using in vivo

pharmacological approaches (aldosterone infusion, MR

antagonism) is hampered by the pleiotropic effects of

Aldo and MR activation, mixing primary events with

the confounding secondary responses, 3) in vitro,

the MR can bind with similar affinities both Aldo and

Gluco. Thus, analyzing the specificity of Aldo/Gluco/

MR signaling pathways is fundamental to understand

their pathophysiological roles in the CV system.

Very few genes have been reported to be modulated

by MR activation in the endothelium, mainly based on

Frédéric JaisserMD, PhD

Research Director - INSERM (French National Institute of Health

and Medical Research).

Paris, France

Contact : [email protected]


short-term Aldo treatment of cultured cells 12, 13, 14.

Activation of MAP kinases pathways has been reported

in vascular smooth muscle cells, as well as induction

of genes involved in vascular fibrosis, inflammation and

calcifications (see for review 15). So far, biomarkers of

MR activation have not been identified.

2- Conditional transgenic models: a clue to understand specific molecular and functional roles of MR activation in the heart and vessels

The profibrotic effect of aldosterone has been

first identified in 1990 by Brilla and Weber in a

pharmacological model combining uninephrectomy,

high salt diet and infusion of aldosterone (or DOCA,

a mineralocorticoid analogue) 16. Both right and left

ventricles presented with interstitial remodelling

suggesting that this was related to humoral disorders

but not hemodynamic alterations due to high blood

pressure. Indeed administration of a low dose of

spironolactone, which was unable to prevent blood

pressure increase, prevented cardiac and vascular

remodelling as well as local inflammation 16-18. This

confirmed that the deleterious effects were not

related to hemodynamic factors but to local Aldo/MR

activation.

Experimental models, such as non-targeted MR gene

inactivation or pharmacological models (manipulating

ligands or MR/GR antagonists) are complex to analyze.

The pleiotropic effects of Aldo and Gluco as well as the

large tissue distribution of their respective receptors

make difficult to unravel their specific contributions to

organ/cell pathophysiology. Inducible transgenic models

allow spatio-temporal control of MR and GR expression,

in selected target cells as well as precise tuning of

receptor expression over time. The targeted approach

rules out the possibility of secondary effects due to

changes in renal ion homeostasis or to general Aldo/

Gluco-induced disturbances and allows investigation on

the specific role of the MR/GR in the cardiomyocytes

or vessels 19. This segmental analysis requires in a

second step an approach with pharmacological or

constitutive, generalized gene manipulations.

Several double-transgenic mouse models with

conditional, inducible manipulation of MR and GR

expression have been generated and functionally

analyzed in our laboratory. Such animal models provide

tools that allowed to progress in the understanding

of the steroid-mediated pathophysiology. This allows

to address: i) Molecular and functional effects of MR

in each target organ/cell type ii) Integrated in vivo

responses permitting the analysis of both primary

and secondary effects iii) Combination of genetic and

pharmacological approaches to define specificity of

the responses and of the identified pathways.

Pathophysiological role of MR/GR in the heart

1- Cardiac MR over-expression led to major

electrocardiographic abnormalities with prolonged

ventricular repolarisation and spontaneous and

triggered ventricular arrhythmias. This was

associated with ion channel remodelling (patch-clamp)

leading to a decrease in Ito K current and an increase in

action potential duration and Ca transient amplitude.

During embryogenesis and adulthood, mice exhibited

a high rate of death prevented by the MR antagonist

spironolactone 20.

2- A cardiac GR over-expressing model shows

several phenotypic differences as compared to the

MR model, with major conduction defects and a mild

dilated cardiomyopathy, but no embryonic lethality.

Ion channel remodelling also differed, indicating that

specific pathways are distinctly regulated via MR or

GR activation in the heart 21.

oVereXpression oF tHe minerAlocorticoid receptor: pAtHopHYsioloGicAl conseqUences

Pathophysiological role of MR in the vessels

In order to manipulate Aldo/Gluco and MR/GR

signalling in the vessels, MR over-expression has

been recently achieved in endothelial cells using an

endothelial-specific transactivator strain. Increased

MR activation in endothelial cells only is associated

with increased blood pressure (independently of

alterations in ion homeostasis), altered vascular

reactivity as estimated ex vivo in mesenteric

arteries and altered in vivo blood pressure

response to vasoconstrictors like AngII and ET1.

Identification of specific gene targets of the MR

Differential screening of gene targets specifically

modulated upon MR activation but not GR activation

was done in the heart of the MR/GR conditional models.

Using transcriptome analysis done in heart samples

from mice with conditional overexpression of either

MR or GR in the cardiomyocytes only, we analyzed

differential expression of 10 000 mouse genes (using

MWG microarrays) as well as 6000 genes selected

for their putative implication in heart diseases (these

genes were selected based on their expression

in cardiac tissue affected by various pathologies).

Some of the genes overlapped between the two

arrays. We were able to identify networks of genes

specifically up- or down- regulated in the MR or GR

model. Several candidates were selected from these

results and validated in individual mice at different

time points after MR/GR expression. About 15 genes

that were up- or down- regulated in the MR model

(as compared to control mice and GR mice) were

validated.

Some of them have been proposed as putative

biomarkers of MR activation since they encodes

secreted protein that have been identified in the

plasma of these transgenic models as well as in a rat

heart failure model. Interestingly, pharmacological

MR antagonism normalized the increased plasma

level of these biomarkers.

Taken together, our data indicate that:

-Our unique models are powerful tools to identify

and validate biomarkers of MR activation.

-Such biomarkers of MR activation may be particularly

useful in human and canine patients with heart failure,

human patients with infarct, metabolic syndrome

(including diabetes), as companion diagnostic in order

to adapt the treatment on an individual basis. Indeed

we have shown that one of these markers is also

expressed in the dog but its relevance in canine heart

failure remains to be studied.


References

1. Farman N, Rafestin-Oblin ME. Multiple aspects of mineralocorticoid selectivity. Am J Physiol Renal Physiol. 2001;280(2):F181-192.

2. Latouche C, Jaisser F. Contribution of transgenesis models in discovery of new pharmacological targets in heart failure: aldosterone receptor as an example.

Therapie. 2009;64(2):81-86.

3. Christy C, Hadoke PW, Paterson JM, Mullins JJ, Seckl JR, Walker BR. 11beta-hydroxysteroid dehydrogenase type 2 in mouse aorta: localization and influence on

response to glucocorticoids. Hypertension. 2003;42(4):580-587.

4. Maron BA, Leopold JA. Mineralocorticoid receptor antagonists and endothelial function. Curr Opin Investig Drugs. 2008;9(9):963-969.

5. Struthers AD. Aldosterone-induced vasculopathy. Mol Cell Endocrinol. 2004;217(1-2):239-241.

6. Pitt B, Remme W, Zannad F, Neaton J, Martinez F, Roniker B, Bittman R, Hurley S, Kleiman J, Gatlin M. Eplerenone, a selective aldosterone blocker, in patients with

left ventricular dysfunction after myocardial infarction. N Engl J Med. 2003;348(14):1309-1321.

7. Pitt B, Zannad F, Remme WJ, Cody R, Castaigne A, Perez A, Palensky J, Wittes J. The effect of spironolactone on morbidity and mortality in patients with severe

heart failure. Randomized Aldactone Evaluation Study Investigators. N Engl J Med. 1999;341(10):709-717.

8. Hostetter TH, Rosenberg ME, Ibrahim HN, Juknevicius I. Aldosterone in progressive renal disease. Semin Nephrol. 2001;21(6):573-579.

9. Takeda M, Tatsumi T, Matsunaga S, Hayashi H, Kimata M, Honsho S, Nishikawa S, Mano A, Shiraishi J, Yamada H, Takahashi T, Matoba S, Kobara M, Matsubara H.

Spironolactone modulates expressions of cardiac mineralocorticoid receptor and 11beta-hydroxysteroid dehydrogenase 2 and prevents ventricular remodeling in

post-infarct rat hearts. Hypertens Res. 2007;30(5):427-437.

10. Ohtani T, Ohta M, Yamamoto K, Mano T, Sakata Y, Nishio M, Takeda Y, Yoshida J, Miwa T, Okamoto M, Masuyama T, Nonaka Y, Hori M. Elevated cardiac tissue level

of aldosterone and mineralocorticoid receptor in diastolic heart failure: Beneficial effects of mineralocorticoid receptor blocker. Am J Physiol Regul Integr Comp

Physiol. 2007;292(2):R946-954.

11. DeLano FA, Schmid-Schonbein GW. Enhancement of glucocorticoid and mineralocorticoid receptor density in the microcirculation of the spontaneously

hypertensive rat. Microcirculation. 2004;11(1):69-78.

12. Leopold JA, Dam A, Maron BA, Scribner AW, Liao R, Handy DE, Stanton RC, Pitt B, Loscalzo J. Aldosterone impairs vascular reactivity by decreasing glucose-6-

phosphate dehydrogenase activity. Nat Med. 2007;13(2):189-197.

13. Caprio M, Newfell BG, la Sala A, Baur W, Fabbri A, Rosano G, Mendelsohn ME, Jaffe IZ. Functional mineralocorticoid receptors in human vascular endothelial cells

regulate intercellular adhesion molecule-1 expression and promote leukocyte adhesion. Circ Res. 2008;102(11):1359-1367.

14. Ducros E, Berthaut A, Mirshahi SS, Faussat AM, Soria J, Agarwal MK, Mirshahi M. Aldosterone modifies hemostasis via upregulation of the protein-C receptor in

human vascular endothelium. Biochem Biophys Res Commun. 2008;373(2):192-196.

15. Viengchareun S, Le Menuet D, Martinerie L, Munier M, Pascual-Le Tallec L, Lombes M. The mineralocorticoid receptor: insights into its molecular and (patho)

physiological biology. Nucl Recept Signal. 2007;5:e012.

16. Brilla CG, Pick R, Tan LB, Janicki JS, Weber KT. Remodeling of the rat right and left ventricles in experimental hypertension. Circ Res. 1990;67(6):1355-1364.

17. Brilla CG, Matsubara LS, Weber KT. Anti-aldosterone treatment and the prevention of myocardial fibrosis in primary and secondary hyperaldosteronism. J Mol Cell

Cardiol. 1993;25(5):563-575.

18. Brilla CG, Weber KT. Mineralocorticoid excess, dietary sodium, and myocardial fibrosis. J Lab Clin Med. 1992;120(6):893-901.

19. Nguyen DC, Sainte-Marie Y, Jaisser F. Animal models in cardiovascular diseases: new insights from conditional models. Handb Exp Pharmacol. 2007(178):377-405.

20. Ouvrard-Pascaud A, Sainte-Marie Y, Benitah JP, Perrier R, Soukaseum C, Cat AN, Royer A, Le Quang K, Charpentier F, Demolombe S, Mechta-Grigoriou F, Beggah

AT, Maison-Blanche P, Oblin ME, Delcayre C, Fishman GI, Farman N, Escoubet B, Jaisser F. Conditional mineralocorticoid receptor expression in the heart leads to

life-threatening arrhythmias. Circulation. 2005;111(23):3025-3033.

21. Sainte-Marie Y, Cat AN, Perrier R, Mangin L, Soukaseum C, Peuchmaur M, Tronche F, Farman N, Escoubet B, Benitah JP, Jaisser F. Conditional glucocorticoid

receptor expression in the heart induces atrio-ventricular block. Faseb J. 2007. 3133-41.

oVereXpression oF tHe minerAlocorticoid receptor: pAtHopHYsioloGicAl conseqUences

qUestion sessionnicolette FArmAn (nF) And Frédéric JAisser (FJ)

AnsWers From:

nicolette FArmAn (nF) md, phd - research director, inserm, paris, France

Frédéric JAisser (FJ) md, phd - research director, inserm, paris, France

Question (Chair) - There is interest in non-genomic effects of mineralocorticoid receptors (MR) –

are these of physiological significance, or simply a red herring?

Answer (NF) - Aldosterone acts rapidly in the cytoplasm to modulate cell function in cell cultures, for

example on calcium and pH, suggesting such effect but these have not been proven in living animals

or humans. So we do not yet know the answer.

Question (Delegate) - How much do we know about MR in fibroblasts? Does activation have an

effect on the amount of collagen, or the cross-linking or quality of the collagen?

Answer (FJ) - High doses of aldosterone have been shown to induce collagen synthesis in fibroblasts

in vitro, but it is not know if MR is expressed in fibroblasts in vivo. Aldosterone has been shown to

bind to glucocorticoid receptors (GR) in many studies and high doses of MR antagonists appear to

block GR activity. This could, therefore, explain how aldosterone induces collagen synthesis. Current

consensus is, however, that fibrosis is not necessarily a direct effect of MR activation and that other

factors, e.g. oxidative stress, may have a secondary effect on remodelling and vascular changes.

Question (Delegate) - How much is known of the different MR genetic variants? And does treatment

with MR antagonists differ between these genotypes?

Answer (NF) - There is a mutation that affects the MR - a single amino acid mutation - that changes the

nature of the MR-aldosterone ligand so that progesterone, for instance, changes from an antagonist

to an agonist. Also, spironolactone becomes an agonist and children with this mutation treated with

spironolactone actually develop hypertension. However, little is known about other mutations and how

they differ in their sensitivity to antagonists.


Question (Delegate) - It is fascinating that, in the RALES study, the effects of spironolactone

were independent of measured circulating concentrations of aldosterone. Why is this? Are there

factors that alter the downstream response to MR, or is there more aldosterone production and

intracellular action that is not reflected in measurements from blood samples?

Answer (FJ) - There is debate and several possibilities: 1) is there local production of aldosterone, i.e.

in cardiomyocytes? 2) Is there increased production of MR? 3) Is there increased capacity of MR to

be activated by the same level of aldosterone, or is there more co-activator produced or available?

4) Is the ligand not aldosterone but glucocorticoid in this clinical setting? Due to oxidative stress,

there is a change in the ability of MR to be activated by glucocorticoids. There is a correlation

between glucocorticoid levels and survival from heart disease, which may be due to activation of GR.

There is, therefore, no clear explanation.

Question (Speaker: Robert Hamlin, USA) - The development of drugs that serve as ligands for many

receptors and which have minimal side effects is desirable. Would it, therefore, be better to

develop drugs that work further downstream, e.g. on calmodulin, and focus on specific end points?

Answer (FJ) - Yes, but will it affect the pathology? I am not sure that such an approach would be

specific enough.

Question (Chair) - How are we going to develop MR antagonists with tissue-specific effects?

Answer (NF) - I don’t know. If I did I would be very rich and not here at the moment! Can we identify co-

activators or co-repressors that are more specific for MR than other receptors? Or tissue-specific

receptors? These are excellent targets for our research and are the future.

Answer (FJ) - You need to bear in mind that antagonists block receptors in certain configurations and

so, dependent upon the cofactor, the structure of the receptor is different.

Question (Delegate) - Can we assume that all dog breeds would have the same MR expression in

cardiomyocytes? After all there is evidence in rats that different levels of MR expression occur

between breeds.

Answer (NF) - In principle it is possible that the expression of MR in the hearts of dogs would mimic

that seen in other species. It is not difficult to do the research to find out.

Comment (Chair) - Pharmacogenomics is a huge area and one in which we are way behind in

veterinary medicine. Especially since dogs represent such a wide range of breeds with hugely

different genetics.


Johann Bauersachs is an Associate Professor at the University Hospital of Würzburg (Germany) in the Division of Cardiology. He has Board Certifications in Internal Medicine Cardiology and Critical Care. He received two Awards from the German Society of Cardiology (2001, 2004) and more recently the Parmley-Award of the American College of Cardiology in 2007.

His current research topics are healing and remodeling after myocardial infarction; cardiac ischemia- and reperfusion injury; cardiac regeneration by progenitor cells; endothelial progenitor cells, endothelial dysfunction and platelet activation in coronary artery disease, myocardial infarction, hypertension and diabetes mellitus.

He is member of the Editorial Board of Cardiovascular Research, European Journal of Clinical Investigation, and Basic Research in Cardiology. Johann Bauersachs is the President of the European Section of the Aldosterone Council (ESAC) in Germany since 2006.

minerAlocorticoid receptor blocKAde: HoW eXperimentAl eVidence sUpports tHe clinicAl beneFit

Activation of the renin-angiotensin-aldosterone

system plays an important role in the pathogenesis

of cardiovascular disease. High levels of aldosterone

impair cardiac and vascular function and predict

mortality risk of patients with acute myocardial

infarction or heart failure. Patients with primary

hyperaldosteronism display a higher incidence of

myocardial infarction and stroke. Large clinical trials

(RALES, EPHESUS) have shown mineralocorticoid

receptor (MR) blockade to decrease mortality in

heart failure 1,2.

In contrast to the classical notion that

mineralocorticoids were only involved in body

electrolyte and water homeostasis mediated by

the kidney, aldosterone exerts important direct

(patho)physiological effects on the cardiovascular

system. While the extra-adrenal generation of

aldosterone from heart and vessels appears to

be of minor importance, MR expression has been

repeatedly documented in cardiovascular cells.

Aldosterone targets MR on cardiomyocytes,

cardiac fibroblasts, endothelial cells and smooth

muscle cells leading to cardiac hypertrophy, fibrosis

and vascular injury. MR expression in the heart is

increased in heart failure explaining detrimental

effects of aldosterone even in the absence of

markedly elevated circulating levels of aldosterone.

Under certain pathophysiological circumstances,

also glucocorticoids may act as agonists on the

MR. Upon binding to the MR aldosterone increases

cardiomyocyte as well as vascular endothelial

and smooth muscle cell reactive oxygen species

formation 3,4,5.

In dogs with chronic heart failure induced by

microembolization, long-term treatment with the

MR blocker eplerenone attenuated progressive left

ventricular (LV) dysfunction as well as LV dilatation,

hypertrophy and fibrosis. In dogs with heart

failure induced by rapid pacing, spironolactone

reduced atrial structural and electric remodelling,

associated with significantly less and shorter

episodes of atrial fibrillation 6,7.

Long-term MR blockade with eplerenone and

angiotensin converting enzyme (ACE) inhibition

alone or in combination were studied in rats with

heart failure after extensive myocardial infarction.

Like ACE inhibition, eplerenone attenuated LV

filling pressure and volume. Combination therapy

significantly improved LV systolic and diastolic

function, and reduced LV end-diastolic volume. In

parallel, molecular alterations such as changes in

collagen expression or myosin heavy chain isoforms

were reversed by combination therapy 8.

In organ baths studies, the effect of MR

blockade on endothelial function was assessed

using phenylephrine-preconstricted aortic rings

from rats with chronic heart failure. Long-term

treatment with spironolactone in addition to

Johann bauersachsMD

Associate Professor, Consultant Cardiology and Critical Care

Department of Medicine I, University Hospital

Würzburg, Germany



ACE inhibition normalized the acetylcholine-

induced nitric oxide-mediated relaxation which

was significantly attenuated in rats treated with

placebo, and prevented the increase in superoxide

anion formation. MR blockade also attenuated the

platelet activation observed in rats with heart

failure, an effect which may also translate into

reduction of thrombo-embolic events in patients

with heart failure 9,10.

To clarify that indeed direct effects in the heart

mediate beneficial actions of MR blockade, we

investigated LV remodeling after myocardial

infarction in mice with cardiomyocyte-specific

deletion of the MR gene (MRMLCCre) which were

generated using a conditional MR allele MRflox

in combination with a transgene expressing Cre

recombinase under control of the myosin light

chain (MLC2a) gene promoter. Sham-operated

wildtype and MRMLCCre mice showed similar cardiac

morphology and function. LV function was preserved

and LV dilatation was attenuated in the MRMLCCre

mice with chronic heart failure compared to

wildtype animals. This was associated with reduced

superoxide formation, myocyte cross-sectional

area and collagen accumulation in the surviving

myocardium. Thus, myocyte-specific deletion of

the MR gene protects against adverse cardiac

remodeling and contractile dysfunction in ischemic

heart failure, suggesting that clinical benefits of

MR blockade in patients with heart failure may

be mediated at least in part via a cardiomyocyte-

dependent mechanism.

As in the EPHESUS study in patients with acute

myocardial infarction complicated by heart failure

there was a striking reduction of mortality by

eplerenone as early as 30 days after randomisation,

we examined whether MR antagonism promotes

healing of the infarcted myocardium. Starting

immediately after coronary ligation, rats were

treated with the selective MR antagonist

eplerenone or placebo. At seven days, eplerenone

therapy versus placebo significantly reduced

thinning and dilatation of the infarcted wall,

improved left ventricular LV function and enhanced

neovessel formation in the injured myocardium 11.

As bone marrow derived endothelial progenitor

cells (EPC) play an important role in endothelial

repair and angiogenesis, we hypothesized that

hyperaldosteronism impairs EPC function and

vascularisation capacity in mice and humans.

Treatment of human EPC with aldosterone induced

translocation of the MR and impaired multiple

cellular functions of EPC, such as differentiation,

migration, and proliferation in vitro. Aldosterone

protein kinase A-dependently increased reactive

oxygen species formation in EPC. Aldosterone

infusion in mice impaired EPC function and

vascularisation capacity in a MR-dependent

manner. EPC from patients with primary

hyperaldosteronism compared to age-matched

controls displayed reduced migratory potential.

MR blockade in patients with hyperaldosteronism

improved EPC as well as endothelial function.

Thus, normalization of EPC function may represent

a novel mechanism contributing to the beneficial

effects of MR blockade in heart failure and other

cardiovascular diseases.

In the Würzburg heart failure registry including

consecutive heart failure patients with either

preserved or reduced LV function, higher serum

levels of both cortisol and aldosterone were

independent predictors of increased mortality

risk conferring complementary and incremental

prognostic value. While numerous studies suggest

that MR blockade is also effective in heart failure

with preserved ejection fraction, definitive evidence

will come from ongoing randomised, placebo-

controlled studies 12.

In conclusion, there is ample evidence from

experimental and clinical trials that mineralo-

corticoid receptor blockade reduces mortality

and morbidity in heart failure.

Low dosages of spironolactone and eplerenone

devoid of substantial diuretic effects have been used

in the large randomised clinical trials. Experimental

evidence supports the concept that direct actions

of MR blockade in the heart and vasculature

mediate at least part of the beneficial effects on

left ventricular remodelling.

minerAlocorticoid receptor blocKAde: HoW eXperimentAl eVidence sUpports tHe clinicAl beneFit

Selected References

1. Pitt, B, Remme, W, Zannad, F, Neaton, J, Martinez, F, Roniker, B, Bittman, R, Hurley, S, Kleiman, J, Gatlin, M (2003) Eplerenone,

a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction. N Engl J Med 348(14): 1309-1321.

2. Pitt, B, Zannad, F, Remme, WJ, Cody, R, Castaigne, A, Perez, A, Palensky, J, Wittes, J (1999) The effect of spironolactone on morbidity

and mortality in patients with severe heart failure. Randomized Aldactone Evaluation Study Investigators. N Engl J Med 341: 709-717

3. Bauersachs, J, Fraccarollo, D (2003) Aldosterone antagonism in addition to angiotensin-converting enzyme inhibitors in heart failure.

Minerva Cardioangiol 51(2): 155-164

4. Bauersachs, J, Fraccarollo, D (2006) Endothelial NO synthase target of aldosterone. Hypertension 48(1): 27-28

5. Lombes, M, Alfaidy, N, Eugene, E, Lessana, A, Farman, N, Bonvalet, JP (1995) Prerequisite for cardiac aldosterone action.

Mineralocorticoid receptor and 11 beta-hydroxysteroid dehydrogenase in the human heart. Circulation 92: 175-182

6. Suzuki, G, Morita, H, Mishima, T, Sharov, VG, Todor, A, Tanhehco, EJ, Rudolph, AE, McMahon, EG, Goldstein, S, Sabbah,

HN (2002) Effects of long-term monotherapy with eplerenone, a novel aldosterone blocker, on progression of left ventricular dysfunction

and remodeling in dogs with heart failure. Circulation 106(23): 2967-2972

7. Yang, SS, Han, W, Zhou, HY, Dong, G, Wang, BC, Huo, H, Wei, N, Cao, Y, Zhou, G, Xiu, CH, Li, WM (2008) Effects of spironolactone on electrical

and structural remodeling of atrium in congestive heart failure dogs. Chin Med J (Engl) 121(1): 38-42.

8. Fraccarollo, D, Schäfer, A, Hildemann, S, Galuppo, P, Ertl, G, Bauersachs, J (2003) Additive Improvement of Left Ventricular Remodeling and Neurohormonal

Activation by Aldosterone Receptor Blockade With Eplerenone and ACE Inhibition in Rats With Myocardial Infarction. J Am Coll Cardiol 42: 1666-1673.

9. Bauersachs, J, Heck, M, Fraccarollo, D, Hildemann, S, Ertl, G, Wehling, M, Christ, M (2002) Addition of spironolactone to ACE inhibition in heart failure improves

endothelial vasomotor dysfunction: Role of vascular superoxide anion formation and endothelial NO synthase expression. J Am Coll Cardiol 39: 351-358.

10. Schäfer, A, Fraccarollo, D, Hildemann, S, Christ, M, Eigenthaler, M, Kobsar, A, Walter, U, Bauersachs, J (2003) Inhibition of platelet activation in congestive heart

failure by selective aldosterone receptor antagonism and angiotensin converting enzyme inhibition. Thromb Haemostas 89(l): 1024-1030.

11. Fraccarollo, D, Galuppo, P, Schraut, S, Kneitz, S, van Rooijen, N, Ertl, G, Bauersachs, J (2008) Immediate mineralocorticoid receptor blockade improves myocardial

infarct healing by modulation of the inflammatory response. Hypertension 51(4): 905-914.

12. Guder, G, Bauersachs, J, Frantz, S, Weismann, D, Allolio, B, Ertl, G, Angermann, CE, Stork, S (2007) Complementary and incremental mortality risk prediction

by cortisol and aldosterone in chronic heart failure. Circulation 115(13): 1754-1761.


qUestion sessionJoHAnn bAUersAcHs

AnsWers From:

JoHAnn bAUersAcHsmd - Associate professor and consultant in cardiology and critical care, University Hospital, Würzburg, Germany

Question (Delegate) - I would like to know how diffuse are the lesions caused by aldosterone over-

activation? Are the lesions concentrated around the main infarction? How about lesions in dilated

cardiomyopathy, for instance? Are these in the left or right ventricles?

Answer - Lesions are not confined to the infarct areas. We have looked at the surviving myocardium and

found marked collagen accumulation and seen effects of MR blockers. It is clear that spironolactone is of

clinical benefit in right-sided heart failure, for example in patients with secondary hyperaldosteronism.

There has been no investigation of fibrosis in humans with right-sided heart failure for the reason

that it is difficult to obtain tissue. I would expect to see less fibrosis in the right ventricle where the

right-sided failure is secondary to left-sided failure or lung disease.

Comment (Speaker: Claudio Bussadori, Italy) - The models discussed are good for looking at systolic

dysfunction in the veterinary field, but there is no real model of chronic volume overload due to mitral

regurgitation, especially in advancing age. If we are really doing something with spironolactone,

we perhaps need myocardial biopsies. To compare experimental mitral valve insufficiency with

spontaneous disease is completely wrong, as the experimental models, which involve cutting the

chordate tendinae, result in acute mitral valve insufficiency. So the experimental model should,

in fact, be the disease itself.

Question (Chair) - Of the human studies involving patients with non-ischaemic heart disease, which

best relate to veterinary medicine?

Answer - These points are well taken and we will see the dog data later. Similar mechanisms work in

different cell types in chronic heart failure because of, for example, this ‘foetal switch’. So, to some

extent, they are all similar at the end stage regardless of how disease has been induced. The situation

is, however, completely different when we want to prevent progression in the earlier stages.

Question (Speaker: Claudio Bussadori, Italy) - Sudden death is a real risk soon after acute myocardial

infarction due to ventricular fibrillation. But this does not happen in chronic mitral valve regurgitation.

One may talk about right ventricular failure due to chronic cor pulmonale, but this is not common in

dogs compared with humans. Emphysema is rare in dogs because they do not live long enough for this

to develop. They probably do develop right ventricular dysfunction because of a dilated left ventricle

and displacement of the septum. In these cases the diuretic effect of spironolactone is important and

therapeutic benefit is not simply due to its antifibrotic effect.

Answer - The best evidence that spironolactone works in humans comes from the RALES study

and here there was no difference between patients with ischaemic or non-ischaemic disease such

as dilated cardiomyopathy. A lot of these patients also had mitral insufficiency and in these cases,

spironolactone still worked extremely well.

Question (Chair) - There is clearly evidence in the literature concerning acute models of myocardial

infarction, some of which involve dogs, but most of the audience here are interested in chronic

heart failure. Which of the experimental models are predictive of benefit in chronic heart failure?

Answer - The mouse and rat myocardial infarction models are not, in fact, models of acute disease.

Heart failure develops within several weeks and so they are viewed as models of chronic heart failure.

For example, much of the development work on ACE inhibitors was done using these models during

the 1980s and early 90s. To be efficient as models of heart failure, we make large infarctions and do

not start treatment too early. The dog model in which heart failure is induced by ventricular pacing

is not often used now because it is not necessarily viewed as a good model of human heart failure.

Question (Speaker: Robert Hamlin, USA) - How are you convinced that the relation between

mineralocorticoids and glucocorticoids (in determining survival for humans with heart failure) is

a cause or effect of heart failure?

Answer - There are good data from treatment studies and MR knock-out mice that the effects of MR

activation develop over time, and that it is early as well as late stage MR activation that contributes to

the pathology. There is also a dose effect in that less MR activation is seen in less severe heart failure.

Question (Speaker: Robert Hamlin, USA) - Do you believe that the mechanism by which heart failure

occurs is important in determining the response to therapy? Does the mechanism of induction

determine the response to treatment?

Answer - There appears to be no clinical distinction in terms of symptoms despite the different underlying

pathologies, but the effects of drugs are important and different. The best treatments are those that

work in multiple models of heart failure. Spironolactone, for example, does work in multiple models.


Robert Hamlin is the Stanton Youngberg Professor of Physiology/Pharmacology, a Professor of Biomedical Engineering and a Professor at the Davis Heart and Lung Research Institute, Ohio State University. He is Scientific Director of QTest Labs. His research interests are safety pharmacology, comparative electrocardiography, cardio-pulmonary interactions in the genesis of signs and symptoms, models of heart failure and arrhythmia, cardiac resynchronization therapy, and pulmonary mechanics.

He was Past President and Chairman of the Board of the ACVIM, Past President of the Central Ohio Heart Association, recipient of an NIH Career Development Award, and received Distinguished Professor and Distinguished Lecture Awards at The Ohio State University. Dr. Hamlin has published over 300 refereed research publications and 18 textbook chapters on cardiovascular medicine, and cardiovascular physiology and pharmacology.

tHe proXimAte cAUse oF HeArt FAilUre: models, remodelinG, And re-remodelinG

Definition and Proximate Cause of Heart failure:

Heart failure is a set of signs and symptoms—a

syndrome—produced by a pathological interaction

among the heart, blood vessels, and their

neuroendocrine control. A failing heart is

characterized by: (1) reduced rate of cycling of

heavy meromyosin heads (the fundamental units of

contraction), (2) reduced rate of hydrolysis of ATP

to ADP and release of energy, (3) reduced slope

(Emax) of the connection of end-systolic points

of pressure-volume loops obtained at varying

preloads, and (4) decreased force or rate of force

development for a given preload. Heart failure has

multiple potential causes including valvular and/or

myocardial disease, infection or toxicity, but most

possess in common an overload of pressure and/

or volume which may alter calcium kinetics (release

and binding), depolarization and/or repolarization,

energy transformation and/or availability, or

alignment of contractile elements. The pathway of

calcium kinetics is as follows:

(1) Abnormal release of calcium from the

sarcoplasmic reticulum through the ryanodine

release channel.

(2) The abnormal release is due to depletion of

calcium in the sarcoplasmic reticulum due

to calcium leaks in diastole, or to abnormal

function of the ryanodine release channel.

(3) The deficiency of sarcoplasmic calcium is due

to faulty entry into the sarcoplasmic reticulum

through the SERCa++/ATPase pump and

abnormal exit of calcium from the cytosol via

the up-regulated Na+/Ca++ exchanger.

Deficiency of ATP is not the proximate cause of

heart failure, rather there is inadequate shuttling

of ATP to the contractile elements.

Models of heart failure:

Models of heart failure are developed to study

the genesis of signs and/or symptoms (i.e.,

pathophysiology), and to investigate therapeutic

interventions. Models are classified by etiology:

genetic modification (e.g., tropomodulin over-

expression), toxic (e.g., cobalt, saponin), infectious

(e.g., Trympansoma cruzi), ischemic (e.g., coronary

embolization), electrophysiological (e.g., heart

block, rapid pacing), electrical ablation (e.g.,

transventricular shock), drug-induced (e.g.,

doxorubicin), pressure overload (e.g., aortic/

pulmonic stenosis), volume overload (e.g., AV shunt,

mitral/aortic regurgitation).

The vast majority of dogs presented with signs of

heart disease are affected by mitral regurgitation,

and are small breed, chondrodystrophic, aged

dogs which may have multiple concurrent

degenerative diseases (e.g., pulmonary fibrosis,

chronic bronchitis, microscopic intramural

robert HamlinDVM, PhD, DipACVIM

(Cardiology/Internal Medicine)Stanton Yougberg Professor of Physiology/

Pharmacology, Professor Biomedical Engineering, Professor Davis Heart and

Lung Research Institute, The Ohio State University Scientific Director, QTest Labs

Columbus, Ohio - USA



myocardial infarction, chronic interstitial nephritis,

lenticular sclerosis, periodontitis). The “Perfect

Model” should be produced rapidly, inexpensively,

have a high yield, mimic the natural state, safe for

the investigator, uniform in severity, controllable,

stable, humane; and be acceptable by the academic

community, pharmaceutical industry, and drug

regulatory agencies. Although acute rupture of

either the mitral valvules or chordae tendinae

mimics acute mitral regurgitation, the disease

as observed in the clinic is mimicked better by

adding rapid-pacing induced heart failure.

The ultimate value of a model is determined by

its predictive value (sensitivity and specificity)

for providing scientific knowledge which can be

extrapolated to the target species. The ultimate

value of a model is determined by its predictive

value (sensitivity and specificity) for providing

scientific knowledge which can be extrapolated to

the target species.

Remodeling:

Remodeling is a “catch-all” term for structural,

biochemical, electrophysiological, physico-chemical,

and genetic changes that occur in an organism

that alters the quality and/or duration of life.

Adaptive (physiologic) remodeling translates to

increased quality and or duration of life whereas

maladaptive (pathologic) remodeling decreases

quality and/or duration of life. Mal-adaptive

remodeling often arises from chronic hemodynamic

loading and/or neurohumoral activation. Examples

of adaptive remodeling are the physiologic

responses to exercise (athletes) or that which

occurs with pregnancy. Adaptive remodeling

is characterized by vascular dilatation, a brief

increase in activation of renin-angiotensin-

sympathetic nervous activity, and lengthening of

action potential duration. Adaptive remodeling

may t r ans i t i o n f r om a compensatory,

physiologic process to a maladaptive one.

Reverse remodeling:

Reverse remodeling, “re-remodeling”, refers to

the return towards normal cardiac structure

or function. Whereas re-remodeling may occur

spontaneously after resolution (e.g., viral

myocarditis), withdrawal (e.g., cisapride) or

correction (e.g., pulmonic stenosis) of etiological

agents, it occurs most frequently in response

to active therapy (e.g., surgical closure of

patent ductus arteriosus, ventricular assist

device, cardiac resynchronization, stem cell

transplantation, combination of ACE inhibitor and

spironolactone). Re-remodeling is assessed using

many of the same indices by which remodeling is

determined and quantified: ventricular volumes,

ejection fraction, sphericity index, myocardial

dysynchrony, arrhythmias, and neuroendocrine

a c t i v a t i o n . Agen ts k nown t o p r omote

re-remodeling are: ACE inhibitors, aldosterone

inhibitors, AT1-antagonists, ß-blockers, blockers

of sarcolemma Na+/H+ exchanger, and possibly

dobutamine. Studies from one laboratory

suggested that ACE inhibitor-increase in

bradykinin in the left ventricle actually accelerated

matrix loss and therefore was ineffective in

treating rats with volume overload due to AV

shunts. However the same group found that

ß-receptor blockers decreased activation of the

renin-angiotensin system, and that this attenuated

Ang II–mediated NE and Epi release into the

heart and circulation. Decreasing left ventricular

angiotensin-converting enzyme expression in the early

phases of volume overload was in turn protective.

Therefore they believe that angiotensin-II

is important in the remodeling process and

should be attenuated pharmacological ly,

but imply that ACE inhibition did not attenuate

angiotensin-II production or activity, or possibly

that, contrary to the putative beneficial

vasodilatory activity of bradykinin, the increase in

bradykinin counteracts the benefits of decreased

angiotensin-II production or activity. They also

showed that ß-receptor blockers protected

ventricular function in dogs with iatrogenic mitral

regurgitation, supporting the well-known beneficial

effect of ß-receptor blockers. Since angiotension-

II promotes release and decrease reuptake of NE,

it is inexplicable why ACE inhibitors which

decrease angiotensin-II should not produce a

similar benefit.



References

Arnold L, Llewellyn-Smith I, and Minson J (1999) Animal models of heart failure: The heart and blood vessels in health and disease.

supplement Australian and New Zealand J Med 29:403-409.

Boldt A, Wetzel U, Weigl J, et al. Expression of angiotension II receptors in human left and right artrial tissue in atrial fibrillation with and without

underlying mitral valve disease. J Am Col Cardiol 2003 42:1785-1792.

Cohn, JN, Ferrari, R, Sharpe, N. Cardiac remodeling--concepts and clinical implications: a consensus paper from an international forum on cardiac remodeling.

Behalf of an International Forum on Cardiac Remodeling. J Am Coll Cardiol 2000; 35:569.

Dell’Italia LJ. The renin-angiotensin system in mitral regurgitation: a typical example of tissue activation. Curr Cardiol Rep. 2002 Mar;4(2):97-103. Review.

Dixon J and Spinale F, Large animal models of heart failure. (2009) Circulation: Heart Failure 2:262-271.

Fujii Y, Orito K, Muto M, Wakao Y. Modulation of the tissue renin-angiotensin-aldosterone system in dogs with chronic mild regurgitation through the mitral valve.

Am J Vet Res. 2007 Oct;68(10):1045-50.

Greenberg, B, Quinones, MA, Koilpillai, C, et al. Effects of long-term enalapril therapy on cardiac structure and function in patients with left ventricular dysfunction.

Results of the SOLVD echocardiography substudy. Circulation 1995; 91:2573.

Groenning, BA, Nilsson,JC, Sondergaard, L, et al. Antimodeling effects on the left ventricle during beta-blockade with metoprolol in the treatment of chronic heart failure.

J Am Coll Cardiol 2000; 36:2072.

Hasenfuss G. Alterations of calcium regulatory proteins in heart failure. Cardiovasc Res 1998;37:279–289.

Kalthof B, Sato N, Iwase M et al. Effects of ryanodine on cardiac contraction. J Mol Cell Cardiol 1995; 27:2111–2121.

Li D, Shinagawa K, Pang L, Leung TK, Cardin S, Wang Z, Nattel S. Effects of angiotensin-converting enzyme inhibition on the development of the atrial fibrillation substrate

in dogs with ventricular tachypacing-induced congestive heart failure. Circulation 2001;104:2608–2614.

Lindner M, Erdmann E, Beuckelmann DJ. Calcium content of the mouse in isolated ventricular myocytes from patients with terminal heart failure.

J Mol Cell Cardiol 1998;30:743–749.

Milliez, P, deAngelis, N, Rucker-Martin, C et al., Spironolactone reduces fibrosis of dilated atria during heart failure in rats with myocardial infarction.

Europ Heart J 2005 26:2193-2199.

Monnet E and Chachques C, Animal models of heart failure: What is new? (2005) Ann Thorac Surg 79:1445-1453.

Muders F and Elsner D, Animal models of chronic heart failure. (1999) Pharmacological Res 41:605-612.

Nemoto S, Hamawaki M, De Freitas G, Carabello BA. Differential effects of the angiotensin-converting enzyme inhibitor lisinopril versus the beta-adrenergic receptor

blocker atenolol on hemodynamics and left ventricular contractile function in experimental mitral regurgitation. J Am Coll Cardiol. 2002 Jul 3;40(1):149-54.

Ono K, Yano M, Ohkusa T et al. Altered interaction of FKBP 12.6 as a cause of abnormal calcium release in a rabbit model of left ventricular cardiac hypertrophy.

J Mol Cell Cardiol heart failure. Cardiovasc Res 2000;48:323–331.

Opie, LH, Commerford, PJ, Gersh, BJ, Pfeffer, MA. Controversies in ventricular remodelling. Lancet 2006; 367:356.

Patten R and Hall-Porter M, Small animal models of heart failure. (2009) Circulation: Heart Failure 2:138-144.

Perry GJ, Wei CC, Hankes GH, Dillon SR, Rynders P, Mukherjee R, Spinale FG, Dell’Italia LJ. Angiotensin II receptor blockade does not improve left ventricular function

and remodeling in subacute mitral regurgitation in the dog. J Am Coll Cardiol. 2002 Apr 17;39(8):1374-9.

Sciarretta S, Paneni F, Palano F, Chin D, Tocci G, Rubattu S, Volpe M.(2009) Role of the renin-angiotensin-aldosterone system and inflammatory processes in the

development and progression of diastolic dysfunction. Clin Sci (Lond) 116:467-77.

St. John SM, Pfeffer MA, Plappert T et al., Cardiovascular death and left ventricular remodeling two years after myocardial infarction.

Circulation, 96:3294-3299, 1997.

Tallaj J, Wei CC, Hankes GH, Holland M, Rynders P, Dillon AR, Ardell JL, Armour JA, Lucchesi PA, Dell’Italia LJ. Beta1-adrenergic receptor blockade attenuates angiotensin

II-mediated catecholamine release into the cardiac interstitium in mitral regurgitation. Circulation. 2003 Jul 15;108(2):225-30. Epub 2003 Jul 7.

Vanoli E, Bacchini S, Panigada S et al, Experimental models of heart failure, (2004) Eur Heart J 6:F7-F15.

Weber K and Brilla C (1991) Patholoical hypertrophy and cardiac interstitium. Fibrosis and renin-angiotensin-aldosterone system. Circulation 83:1849-1865.

Yamabe H, Itoh K, Yasaska Y, Takata T, and Yokoyama M. The role of cardiac output response in blood flow distribution during exercise in patients

with chronic heart failure. Eur Heart J 16: 951–960, 1995.


qUestion sessionrobert HAmlin

AnsWers From:

robert HAmlin dVm, phd, dipAcVim – stanton Youberg professor of physiology/pharmacology, professor biomedical engineering, professor davis Heart and lung research institute, the ohio state University, columbus, ohio, UsA

Question (Chair) - The kidney is clearly important in heart failure and there is a relationship between renal disease and heart disease in humans. Do you believe that renal disease plays a role in heart disease in veterinary patients?Answer - Knowing the effects of ACE inhibitors on the heart, I am very enthusiastic about these drugs. My renal friends say that the best thing in their field in the last 20 years is ACE inhibitors. These drugs clearly have value in the treatment of heart and kidney disease, and may be useful in multiple diseases. Specific to your question, one aspect to heart failure is abnormal renal function. We understand the mechanisms by which this occurs and also understand that ACE inhibitors, spironolactone and, less so, diuretics should be useful. Let’s consider diuretic more. There is no argument that if you want to reduce pulmonary oedema there is nothing better than diuretics. But humans do not do as well on high doses of diuretics, so it is important to assess the interaction of the heart, lungs and kidneys when discussing pharmacological approaches.

Question (Delegate) - On your last slide you listed the three active agents that you like (ACE inhibitors, mineralocorticoid receptor antagonists, and ß blockers). Would you consider drugs that improve calcium cycling like pimobendan or levosimendan do belong to that list? Answer - This is very difficult. There is no argument that levosimendan and pimobendan have beneficial effects in the veterinary clinic, but there is a group within the USA that has killed the idea that inotropic agents are beneficial. The issue is whether our responsibility is to the patient or the client. Regulatory agencies and academic bodies should ask whether drugs simply lengthen life, or change the quality of life. Those that use pimobendan know it improves quality of life. They have been shown to reduce lifespan in humans. But they improve life in animals. I doubt that a positive inotrop will ever be approved in human medicine. The reason that I like calcium modifying agents is not necessarily because of their inotropic effects but rather because of their lusitropic actions.

Question (Delegate) - There are several works relating to aldosterone receptor blockers in dogs which show that they do not prevent an increase in ventricular size or systolic dysfunction, but do improve neuroendocrine and electrophysiological function. Is this how these drugs improve cardiac function and outcome?Answer - Absolutely. I believe that the beneficial effects of these drugs are not an acute phenomenon; it takes a while to exert an effect due to interactions rather than immediate direct effects on the heart and blood vessels. I am not disturbed by the absence of an acute effect. But I do expect a chronic effect, which is difficult to analyse. Evidence will, I believe, show that attacking the neuroendocrine basis in veterinary medicine will accrue the same benefits as in humans.


Bertram Pitt is Professor of Medicine Emeritus at the University of Michigan School of Medicine. He is diplomate of the American Board of Internal Medicine and of the American Board of Cardiology.

He is a member of several professional societies such as the American College of Cardiology, the American Society for Clinical Investigation, the American Physiological Society – Circulation Group, the American Federation for Clinical Research and the American Heart Association. He has received awards like the Forest Dewey Dodrill Award for Excellence in 2001 and the James B. Herrick Award in 2005 (both from the American Heart Association).

He has published more than 500 papers in the most important peer-reviewed journals dealing with cardiovascular diseases (Circulation, American Heart Journal, Journal of the American College of Cardiology, Hypertension, European Heart Journal…). He is the first author of RALES and EPHESUS and he is currently leading large scale trials investigating the clinical benefit of mineralocorticoid receptor blockade in cardiac patients.

AFter rAles, tHe JoUrneY continUes

Mineralocorticoid receptor blockade (MRB) is

emerging as an important component of the

therapeutic approach to heart failure (HF). The

results of the RALES trial 1 showing a 30% reduction

in all cause mortality in patients with chronic

severe HF (NYHA class III-IV) due to systolic left

ventricular dysfunction (SLVD) randomized to the

MRB spironolactone supported by the results of

the EPHESUS trial 2 in patients with HF and SLVD

post myocardial infarction randomized to the MRB

eplerenone has led to a class 1 indication for MRB

in both European and US guidelines. The benefits

of MRB on mortality in HF appear to be in addition

to standard therapy including an angiotensin

converting enzyme inhibitor (ACE-I) or angiotensin

receptor blocker (ARB), a beta adrenergic receptor

blocker (BB), a loop diuretic and digoxin.

Spironolactone at 12.5-50 mg/day has been shown

to be effective in reducing mortality in the RALES

trial. This beneficial effect has been shown to be

related to a reduction in myocardial and vascular

inflammation, fibrosis, and hypertrophy as well

as to an improvement in nitric oxide availability.

The effectiveness of MRB in HF can in part be

attributed to the finding that MR expression is

increased in HF 3. Activation of the MR, whether

by aldosterone or cortisol 4, has been shown to

result in an up regulation of myocardial calcium

channel expression, electrical remodeling of the

myocardium, and a propensity toward sudden

cardiac death as well ventricular remodeling and

death due to progressive HF.

Elevated levels of both plasma aldosterone and

cortisol have been shown to predict an increase

in mortality and morbidity in patients with chronic

HF 5. Of interest is the recent finding that plasma

aldosterone levels in the upper tertile, but within

normal limits, are predictive of an increase in

mortality and morbidity in patients post myocardial

infarction independent of the presence of HF,

suggesting an important role of MRB in patients

post myocardial infarction without HF or SLVD 6.

MRB is currently being evaluated in patients with

NYHA Class II HF due to SLVD in the EMPHASIS-HF

trial 7 and in patients with HF and a normal left

ventricular ejection fraction (HFNEF) (Diastolic HF)

in the TOPCAT trial 8. In view of the finding that

MRB prevents myocardial and vascular fibrosis

and hypertrophy it can be postulated that in the

future MRB will play an even more important role

in the prevention of HF in patients with essential

hypertension and in the treatment of the entire

spectrum of HF.

bertram pitt MD

Professor of Medicine Emeritus, University of Michigan,

School of Medicine,

Ann Arbor, Michigan, USA



References

1. Pitt, B. et al. The effect of spironolactone on morbidity and mortality in patients with severe heart failure.

Randomized Aldactone Evaluation Study investigators. N Eng J Med 1999, 341: 709-717

2. Pitt, B. et al. Eplerenone, a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction.

N Engl J Med 2003, 348 (14): 1309-1321

3. Yoshida, M. et al. Mineralocorticoid receptor is over expressed in cardiomyocytes of patients with congestive heart failure.

Congest Heart Fail 2005; 11: 12-16

4. Funder, JW. RALES, EPHESUS, and redox. J. Steroid Biochem Mol. Biol. 2005; 93: 121-125

5. Gruder, G. et al. Complementary and incremental mortality risk prediction by cortisol and aldosterone in chronic heart failure.

Circ. 2007; 115: 1754-1761

6. Palmer, BR. et al. Plasma aldosterone levels during hospitalization are predictive of survival post-myocardial infarction.

Eur. Heart J. 2008; 29: 2489-2496

7. Clinical Trials. Gov. Emphasis –HF NCT00232180

8. Aldosterone antagonist therapy for adults with heart failure and preserved systolic function.

Available at clinical trials. Gov. 2007

AFter rAles, tHe JoUrneY continUesqUestion sessionbertrAm pitt

AnsWers From:

bertrAm pitt md – professor of medicine emeritus, University of michigan school of medicine, michigan, UsA

Question (Chair) - One of the key differences in dogs is that, in European markets, a lower dose

of spironolactone is used than in the USA. Have you observed a dose-dependent effect in RALES

and your other studies?

Answer - I am nervous about the dose of spironolactone, especially when it is in the region of 100-

200 mg/day. At the start of RALES we performed a dose-response study, looking at 12.5-100 mg/

day, and found a marked increase in hyperkalaemia at doses in excess of 50 mg/day. Since we were

concerned about drop-outs from the study we settled on a dose of 25 mg/day. There is benefit from

higher doses but you must monitor safety by measuring plasma potassium concentrations, especially

when doses are 100-200 mg/day. There is little diuretic effect below 100 mg/day and we are often

using 15-25 mg once daily or 25 mg every second day.

Question (Speaker: Robert Hamlin, USA) - Comparing the number of persons or animals in heart

failure with the number at risk or in early stages, is there an argument for instituting treatment

very early in the disease?

Answer - Our goal should be to prevent heart failure in humans, but it will take 10-15,000 patients

and several hundred million dollars to perform the requisite clinical studies, and so it is unlikely that

this research will be done.

Question (Chair) - Many veterinary cardiologists are aware of the Alabama group’s model*

looking at acute mitral regurgitation induced by chordae rupture and which has shown that ,at

least in this model, early treatment with an ACE inhibitor prevents fibrosis from occurring and

accelerates the amount of eccentric hypertrophy. Is there any sort of similar caution in too much

neurohormonal blockade too early in human medicine?

Answer – It has not been examined so far in humans with chronic mitral regurgitation. Perhaps it is

something we should go back and look at. I would not use an acute chordae rupture model since the

disease entity is not adequately challenging. In chronic mitral regurgitation, the clues that we know

about in human medicine suggest that ACE inhibitors would be beneficial.

*Dell’Italia’s team, Department of Medicine, Division of Cardiology, University of Alabama at Birmingham, Birmingham, Alabama, and Dillon’s team, Auburn University College of Veterinary Medicine, Auburn, Alabama.

32 - 1st Human and Veterinary Crossover Symposium on Aldosterone

Comment (Speaker: Johann Bauersachs, Germany) - In the CONSENSUS II study, where ACE

inhibitors did not work, men experienced greater mortality in an acute decompensation situation,

suggesting that ACE inhibitors should not be used in this situation.

Comment (Chair) - Your point is very well taken. For veterinary cardiologists, we have always

looked at that models somewhat suspiciously. But we looked at that model because there’s really

nothing else out there in the literature that comes close to approximating what we see in the

chronic situation.

Comment (Delegate) - What the Alabama’s group has been able to show in its experimental dogs

with chordae sectioning is a kind of basic fundamental process showing how a heart dilates and

hypertrophies. It is also important to remember that the category of dogs we see in the clinic are

old. They have vascular changes which has been convincingly shown by Torkel Falk and Lennart

Jönsson. It is a reality and that’s something not seen in these young experimental dogs. There

are probably additional layers than just the fundamental pathophysiology that come into play

on this. These vascular changes do promote a replacement fibrosis that will occur close to the

vessels in the myocardium.

Question (Chair) - Is there any difference between eplerenone and spironolactone?

Answer – I have asked Pfizer many times to do a comparison.There have been a few clinical comparisons

and lots of experimental work. These show that their efficacy is similar. But their safety is different

due to the risk of gynaecomastia in men and premenstrual problems in women being treated with

spironolactone. In primary hyperaldosteronism, spironolactone looks better. Eplerenone has a shorter

half life, which might make it safer.

Question (Delegate) - If you stratified patients according to renal function, would you see any

differences in efficacy of MR antagonists?

Answer – There would be difference in safety, with patients having a GFR <60 being more at risk of

hyperkalaemia. There may be an efficacy difference if the GFR is less than 50 or 30, but there is little

evidence of spironolactone efficacy in these patients probably because of risk of hyperkalaemia. That’s

about all I can say; there simply isn’t yet a firm answer to your question.


Claudio Bussadori is Director of the Gran Sasso Veterinary Clinic and Researcher at the paediatric cardiology department of S.Donato Hospital in S.Donato Milanese. He graduated as a Medical Doctor in 2001, thus becoming a human physician in addition to veterinary surgeon.

He is Director of the ECVIM (European College of Veterinary Internal Medicine, Cardiology) Residency programme in his clinic and he also gives seminars lecture on congenital heart diseases at Faculty of Medicine, University of Milan.He was Vice President of the ECVIM between 1993 and 1999, ESVC President from 1997 to 1999. He has been an Honorary Member of the ESVC Board since 2002.

His main fields of interest are diagnosis, epidemiology and interventional treatment of congenital heart diseases in dogs; echocardiography and pathophysiology of congenital cardiac diseases in humans and application of new echocardiographic techno log ies (2D Stra in and 3D echocardiography) in human and veterinary cardiology.

eFFicAcY oF spironolActone in doGs WitH nAtUrAllY occUrrinG mYXomAtoUs mitrAl VAlVe diseAse

1.Introduction

In the RALES study (Randomized ALdactone

Evaluation Study) a 31% reduction in the risk of

mortality (cardiac causes) in patients receiving

Spironolactone led to the premature termination of

this study for ethical reasons. Aldosterone is thought

to be similarly involved in the pathophysiology of canine

heart failure (HF), hence clinical field studies were

conducted to test whether Spironolactone would

also be beneficial in dogs. The aim of these trials

was to assess the effect of Spironolactone therapy

on the risk of sudden cardiac death, euthanasia for

cardiac reasons, or worsening of HF when compared

to placebo in dogs with moderate to severe mitral

regurgitation (MR) caused by MMVD (myxomatous

mitral valve disease).

2.Materials and Methods

Animals

221 dogs were enrolled from 32 practices in

France, Germany, Belgium and Italy, between

February 2003 and March 2005. The study was

completed in May 2006.

Study design

This multicenter study was a prospective, double-

blinded, placebo-controlled, randomized study.

The complete clinical trial process was conducted

according to Good Clinical Practice (GCP). Owner

consent was obtained with the option to withdraw

the dog at any time.

The follow-up comprised two consecutive stages.

At the first stage, dogs were recruited in 2

separate studies: one 2-month study, where

Furosemide treatment was mandatory at inclusion,

and one 3-month study, where Furosemide was not

allowed at the time of inclusion.

The second stage was a 12-month study involving

dogs that had completed either of the first

studies. Dogs which completed the first studies

were entered into the 12-month study, where they

continued to receive the same trial treatment.

Inclusion criteria

At initiation of the first stage, dogs of any breed

or gender were enrolled when they presented with

moderate to severe MR caused by MMVD (ISACHC

class II and class III). Included dogs with dilated

cardiomyopathy (DCM), were excluded from

analyses (see results section). Dogs must

furthermore have presented at least 3 of the

following clinical signs, including at least one of

cough, dyspnoea, syncope and at least one of

reduced activity, reduced mobility, altered demeanor.

All dogs were receiving an ACEI (angiotensin

converting enzyme inhibitor). Furosemide therapy

was prohibited at inclusion for the 3-month study

and mandatory at inclusion for the 2-month study.

claudio bussadoriDVM, MD,

DipECVIM (Cardiology), PhDDirector of Clinica Veterinaria Gran Sasso

Researcher at paediatric cardiology department of S.Donato Hospital

S.Donato Milanese

Milan, Italy



All dogs which had completed one of the initial two

studies could be enrolled in the 12-month study,

if agreed by the owner.

Exclusion criteria

Excluded dogs were those receiving cardiac

medications other than ACEI, Furosemide, Digoxin

and L-carnitine, those with acute pulmonary edema,

a congenital cardiac disease or a life threatening

arrhythmia, or any other diagnosed medical

condition or those treated with any drugs which

could interact with the assessment of the tested

product efficacy (e.g. ß-blockers, calcium channels

inhibitors). In absence of safety data, pregnant

females were not enrolled.

Randomization and blinding conditions

Double-blinding conditions were maintained

throughout the two stages of follow-up for

investigators, owners and study monitors. Dogs

kept the same study number throughout studies

and so were maintained in the same treatment

group: no re-allocation was carried out.

Treatment

All dogs received conventional therapy including at

least an ACEI. Furosemide was allowed after D5

in the 3-month study and from at least one day

before inclusion in the 2-month study. During the

follow-up, veterinarians could change the dose rate,

initiate or terminate Furosemide treatment. Therapy

with Digoxin and/or L-carnitine was also allowed. In

addition, dogs received either Spironolactone (2 mg/

kg once a day with food) or a placebo.

Evaluation schedule

In both f irst studies, examinations had a

similar schedule. Fu l l c l in ica l examinat ion,

thoracic radiography, electrocardiography, echo-

cardiography, urine and blood samples were

performed on Day 0. Clinical and radiographic

examinations and blood sampling were performed

during the first week, at Day 28 (except

radiographs), Day 56 and Day 84. In the long

term study, clinical and radiographic examinations

and blood sampling were performed at 3-month

intervals. Echocardiographic exams were

undertaken at inclusion and after 6 and 12 months.

Clinical evaluation

The protocol included assessment of the clinical

variables cough, dyspnea, exercise intolerance

(outside mobility, activity at home, attitude at

the veterinary practice) and syncope. Lateral and

dorsal thoracic radiographs were used to evaluate

the heart size (using the Buchanan Vertebral Heart

Scale)2 and the presence of pulmonary edema.

Standard echocardiography measurements were

performed. Classification of the stage of heart

failure at inclusion was assessed according to the

International Small Animal Cardiac Health Council

classification (ISACHC)6.

Survival evaluation

The primary end-point was cardiac related death,

euthanasia due to MR, or severe worsening of

MR, which was defined as the need to introduce

an unauthorized cardiac therapy or to increase the

dose of Furosemide over 10 mg/kg/day to prevent life

threatening CHF (congestive heart failure). Where

the dog died spontaneously or was euthanized, the

investigator specified whether the cause of death

was cardiac or non-cardiac and noted the precise

reason. Cardiac mortality was assessed by pooling

natural deaths and euthanasia owing to cardiac

causes.


Statistical analyses

All analyses were two-tailed. The descriptive analysis

and initial comparability between treatment groups

were made on the characteristics and clinical

criteria recorded at inclusion in the two first

studies. The baseline data were compared

between treatment groups, to check that owner

withdrawals had not produced bias, and also

between these two studies (study effect).

The percentage of morbidity-mortality or mortality

events at the end of follow-up was compared

between groups using Fisher’s exact test.

Survival curves were obtained by the Kaplan-Meier

method. The survival analysis was performed by

log rank test, for comparing the survival of the

two treatment groups, and by a multifactorial Cox

proportional hazard model to assess the impact

of some covariates. The hazard ratio and its 95%

confidence interval were also evaluated. Level

of significance was set at p<0.05. Values were

reported as mean ± standard deviation.

3.Results

Baseline characteristics of dogs at inclusion

221 dogs were enrolled. 7 dogs with DCM were

excluded from further analysis, 2 dogs were

lost before the first examination, which left 212

dogs with MMVD (Intention To Treat population).

The baseline data were comparable between the

2 treatment groups. Because of different study

protocols of the two short-term studies (regarding

the use of Furosemide), there were significant

differences between the studies, but those did not

produce any differences between treatment groups.

123 of the 179 dogs which completed the first

studies continued into the 12-month study. The

remaining 56 cases (well-balanced between the

2 groups) were not enrolled because the owners

were reluctant to adhere to the re-examinations in

the 12-month study.

Overall outcome

Effect of therapy on outcome

In the Spironolactone and control groups

respectively, 34.3% and 40% of dogs completed

the 15-month period. In the Spironolactone group,

10.8% of dogs reached the primary end-point and

25.5% in the control group (Fisher’s exact test,

p=0.0046). Causes of withdrawals not related to

HF included the owner’s wish to stop after the

first studies, concomitant disease, the owners

moved away and a car accident.

The estimated 15 months survival rate was 84% for

the Spironolactone dogs and 66% for the control

dogs (Log rank test, p=0.017). If only mortality

(cardiac death and euthanasia related to MR) is

considered, the 15 months survival rate was 92%

in the Spironolactone group and 73% in the control

group (Log rank test, p=0.0071).

Cox Proportional Hazard models

The hazard ratio of treatment effect was 0.45

(95% confidence limits (CL) [0.22-0.90], p=0.023).

This represents a 55% reduction in the risk of

morbidity-mortality (severe degradation, natural

death, or euthanasia related to MR). If only

mortality (natural death and euthanasia related

to MR) was considered the hazard ratio was

0.31 (95% CL [0.13-0.76], p=0.011) which

represented a significant 69% reduction in the risk

of mortality.

The study of origin (2-month study or 3-month

study) and the duration of cardiac treatment

before Day 0 did not affect the estimate of

the hazard ratio of the treatment effect, its


confidence interval, or its statistical significance.

The administration of Furosemide (well balanced in

both treatment groups) significantly reduced the

survival probability. It did not affect the estimate of

the hazard ratio of the treatment effect.

4.Study findings

This study demonstrated that the addition of

Spironolactone to conventional cardiac therapy

significantly reduces the risk of cardiac morbidity

and mortality in dogs with MMVD when compared

to conventional therapy alone (i.e. ACEI plus

Furosemide or Digoxin, i f needed). A 55%

reduction in the risk of cardiac morbidity-mortality

was found. A greater 69% reduction in the risk

of mortality was found when only cardiac mortality

was analyzed.

Analysis of the covariate “Furosemide” in the

multifactorial Cox proportional hazard model was

associated with a significant negative effect

although the beneficial effect of Spironolactone

was not affected. One likely explanation could be

that dogs receiving Furosemide had more advanced

disease at study inclusion.

The baseline data and survival analyses indicate

that the included population consisted of 89.6%

of MMVD-cases starting the study at ISACHC

class II. Hence, the estimated survival rate

(morbidity-mortality) is 66% in our control group

at 15 months. The rate of premature withdrawals

for cardiac reasons (severe deterioration and

death) is 25.5% in our control group at 15 months.

The number of patients reaching the endpoints

in the present study is less than 50%, due to

dogs withdrawn for other reasons (predominantly

owner’s wish to stop after the first studies).

This does not allow the calculation of the median

survival times but anyway the hazard ratio (HR),

which compares the chance that a member of each

population will have an event at any given time, is

the best way to estimate the difference between

two survival curves.

In the present study, all dogs were receiving an

ACE inhibitor, yet Spironolactone caused a 55%

reduction in risk of cardiac death, euthanasia

because of MR or worsening of MR during a

15-months follow up, demonstrating that the

benefit of Spironolactone treatment is additional to

that derived from the use of ACE inhibitors alone.

Considering the reduction in the risk of mortality

from cardiac causes, the results in dogs were even

more marked with a 69% reduction at 15 months

(compared to the 31% reduction observed at

3 years in the RALE Study in human medicine7).

The beneficial effects of Spironolactone on survival

may not only be explained by the diuretic effect

of the drug. In rats1-8-10-12 and human3-5 patients

aldosterone induces myocardial and perivascular

fibrosis and alters the endothelial function of

vessels.9-11 Studies performed in human patients

with CHF showed that these effects are

counteracted by aldosterone antagonists11-13.

Recent literature demonstrated that a proportion

of dogs with naturally occurring MMVD had

intramyocardial arterial changes which were

associated with areas of fibrosis in the

myocardium, so called replacement fibrosis4.

The same authors suggest that more severe

intramyocardial arteriosclerotic changes and more

severe replacement fibrosis shorten the survival

time from the onset of cardiac therapy to cardiac

death or euthanasiaa. We believe that the observed

beneficial effect of Spironolactone on survival time

in the present study could partly be related to

a counteractive effect of Spironolactone on the

arterial changes and the replacement fibrosis.


The small size of the DCM population did not permit a

dedicated analysis. The benefit of Spironolactone in

this pathology should be more deeply investigated.

In conclusion, this clinical trial concerning

Spironolactone therapy over a 15-month period in

dogs with moderate to severe MR caused by MMVD

demonstrated a beneficial effect of Spironolactone

when added to conventional therapy. The risk of

cardiac related death/euthanasia was reduced by

69%, as compared to conventional therapy alone

(i.e., ACEI plus Furosemide or Digoxin, if needed).

This finding supports Spironolactone as part of the

treatment protocol in dogs with MMVD.

References

1. Blasi ER, Rocha R, Rudolph AE, et al. Aldosterone/salt induces renal inflammation and fibrosis in hypertensive rats. Kidney Int 2003; 63(5):1791-800.

2. Buchanan JW, Bucheler J. Vertebral scale system to measure canine heart size in radiographs. JAVMA 1995; 206:194-199

3. Duprez DA, De Buyzere ML, Rietzschel ER, et al. Inverse relationship between aldosterone and large artery compliance in chronically treated heart failure patients.

Eur Heart J 1998; 19 (9): 1371-1376

4. Falk T, Jönsson L, Olsen LH, et al. Arteriosclerotic changes in the myocardium, lung, and kidney in dogs with chronic congestive heart failure

and myxomatous mitral valve disease. Cardiovasc Pathol 2006; 15 (4): 185-193

5. Farquharson CA, Struthers AD. Aldosterone induces acute endothelial dysfunction in vivo in humans: evidence for an aldosterone-induced vasculopathy.

Clin Sci (Lond) 2002; 103 (4): 425-431

6. Fox PR, Sisson D, Moise NS. Textbook of canine and feline cardiology:Principles and clinical practice, 2nd ed. Philadelphia, PA: WB Saunders; 1999; 883-902

7. Pitt B, Zannad F, Remme W J, et al. The effect of Spironolactone on morbidity and mortality in patients with severe heart failure.

N Engl J Med 1999; 341 (10):709-717

8. Pu Q, Neves MF, Virdis A, et al. Endothelin antagonism on aldosterone-induced oxidative stress and vascular remodeling. Hypertension 2003 ; 42 (1) :49-55

9. Rocha R, Williams GH. Rationale for the use of aldosterone antagonists in congestive heart failure. Drugs 2002; 62 (5):723-731

10. Sun Y, Zhang J, Lu L, et al. Aldosterone-induced inflammation in the rat heart : role of oxidative stress. Am J Pathol 2002; 161 (5):1773-1781

11. Shieh FK, Kotlyar E, Sam F. Aldosterone and cardiovascular remodelling: focus on myocardial failure. J Renin Angiotensin Aldosterone Syst 2004; 5(1):3-13

12. Virdis A, Neves MF, Amiri F, et al. Spironolactone improves angiotensin-induced vascular changes and oxidative stress. Hypertension 2002; 40:504-510

13. Zannad F, Alla F, Dousset B, et al. Limitation of excessive extra-cellular matrix turnover may contribute to survival benefit of Spironolactone therapy in patients with

congestive heart failure: insights from the randomized aldactone evaluation study (RALES). Rales investigators. Circulation 2000; 102: 2700-2706.

Endnotes

a. Falk T, Jönsson L, Olsen LH, et al. Correlation of cardiac pathology and clinical findings in dogs with naturally occurring congestive heart failure.

Proceedings of 17th Annual ECVIM Congress, Budapest 2007.


Martin Bland is Professor of Health Statistics in the Department of Health Sciences, University of York. He leads and teaches an M.Sc. module in Clinical Biostatistics and leads and jointly teaches a module on Measurement in Health and Disease. He also contributes sessions to M.Sc. modules in Research Methods, Biostatistics in Research Practice, and Systematic Reviews. He is involved in short course on Clinical Trials, Statistics for Clinical Trials, and Epidemiology and Statistics.

He is particularly interested in the design and analysis of studies of medical measurement and in cluster sampling in both clinical trials and observational studies.He was involved in several clinical trials in human medicine such as ICSS, a comparison of angioplasty and stenting with surgery for the treatment of occlusions of the carotid artery.He is on the editorial board of Statistical Methods in Medical Research. He is author of An Introduction to Medical Statistics and Statistical Questions in Evidence Based Medicine and together with Douglas Altman writes the Statistics Notes series in the British Medical Journal.

principles oF sUrViVAl AnAlYsis illUstrAted bY ceVA’s spironolActone triAls

Time to event data

In health care research we often measure the time

which elapses until some event occurs. We call such

data time to event data. Sometimes the event is

adverse, such as death, sometimes it is beneficial,

such as healing. Because of the examples of time

to event data which were first studied, such data

are often know as survival or failure time data.

The terminal event, death, healing, etc. is called

the endpoint. The statistical techniques developed

to deal with them are known collectively as survival

analysis.

The analysis of time to event data would not require

any special methods if we knew the time to event

of every subject. What makes time to event data

difficult to analyse is that often we do not know the

exact survival times of all cases. Some subjects

will still be surviving when we want to analyse

the data. For some events, such as conception

or readmission to hospital, the event may never

happen for some subjects. Furthermore, when

participants have entered the study at different

times, some of the recent entrants may be

surviving, but have been observed for a short time

only. Their observed survival time may be less than

those participants admitted early in the study

and who have since experienced the event. When

we know for some subjects only that the time to

the event is greater than some value, we say that

the data are censored. This also known as being

withdrawn from follow-up.

Here we shall use the example of the CEVA Santé

Animale trials of Spironolactone for the treatment

of heart failure in dogs. This was a double-blind

randomised trial comparing Spironolactone against

placebo (the Control group). The event was death

or euthanasia because of heart failure and the

time to death or euthanasia was recorded in

days. Censoring occurred because some animals

had not died and were still alive at the time of

analysis, because some dogs were withdrawn from

trial at owners’ request, usually because they no

longer wished to have the regular assessments of

the dog, and because some dogs died from other

causes, such as road traffic accidents. Data are

for 15 months of follow-up.

Kaplan Meier survival estimates

Some censored times may be shorter than some

times to events. We overcome this difficulty by the

construction of what we call a life table. We follow

a hypothetical cohort from any time point onwards.

I shall show how this works through the example.

John martin blandPhD, MSc, DIC,

ARCS, FSS, Hon. MRCR Professor of Health Statistics,

Dept. of Health Sciences, University of York

York, England

Contact: [email protected] Web site: http://martinbland.co.uk/


Table 1. Control group, time to death (days)

6 D 56 C 84 C 92 D 335 D 419 C 450 C 450 C 7 C 56 C 84 C 121 D 354 C 419 C 450 C 450 C 8 D 56 C 84 C 126 C 357 C 420 C 450 C 450 C 10 D 56 C 84 C 154 D 358 C 427 C 450 C 450 C 11 D 57 C 84 C 187 D 369 D 433 C 450 C 450 C 12 D 57 C 84 C 196 D 380 C 436 C 450 C 13 C 57 C 84 C 196 C 389 C 441 C 450 C 20 C 58 C 84 C 207 D 393 C 450 C 450 C 27 D 61 C 85 C 210 C 393 C 450 C 450 C 29 C 63 C 86 C 239 D 400 C 450 C 450 C 47 D 63 D 86 C 244 C 404 C 450 C 450 C 51 D 64 D 87 C 262 C 410 C 450 C 450 C 53 C 66 C 88 D 268 D 411 C 450 C 450 C 55 C 79 C 89 C 268 C 413 C 450 C 450 C 55 C 80 C 91 C 325 D 413 C 450 C 450 C

D = Died C = Censored The table shows time to either death or censoring.

Table 1 shows the survival times for the Control

group. Each time is marked ‘D’ for a heart failure

death at that time and ‘C’ if the data were

censored. Thus, in the first column, the first time

was 6 days and the dog died. The second time was

7 days and the dog was censored.

The start of the calculation is set out in Table 2. For

each time when an event or censoring occurs, we find

the number of dogs who were present at that time,

called the number at risk. There were 110 dogs at

the start. We find the number who died. For the first

time when an event took place, 6 days, there was

one death, leaving 110 – 1 = 109 dogs who did not

die. This is the number surviving to the end of day 6.

We then calculate the proportion who survived to the

next time. At 6 days, this is 109/110 = 0.9909091.

This is the proportion of those starting day 6 who

survived to the end of day 6. On day 7 there is a

censoring, but no deaths. The proportion of dogs

surviving is 1.0000000, and the total survival from

the beginning is unchanged. If we multiply 0.9909091

by 1.0000000 we still have 0.9909091. However,

there is an effect, because the number of dogs who

were observed at the start of the next day has been

reduced by one. On day 8, when there was a death,

there were only 108 at risk, not 109. The proportion

surviving day 8 is 107/108 =0.9907407. We get the

proportion surviving from the beginning of the life

table to the end of day 8 by multiplying the proportion

surviving up to day 8 by the proportion surviving on

day 8: 0.9909091×0.9907407 = 0.9817340.

Now this is not the same as the proportion known

to have survived beyond day 8 from the beginning,

which would be 107/110 = 0.97272727. We have

adjusted the estimate for the dog who was censored

on day 7. This dog contributed information for the

time in which it was observed and we have included

this information in the analysis.

Table 2. Calculation of the Kaplan Meier survival estimates for the Control group (beginning only)

t C D n d s p P 6 0 1 110 1 109 0.9909091 0.9909091 7 1 0 109 0 109 1.0000000 0.9909091 8 0 1 108 1 107 0.9907407 0.9817340 10 0 1 107 1 106 0.9906542 0.9725589 11 0 1 106 1 105 0.9905660 0.9633838 12 0 1 105 1 104 0.9904762 0.9542087 13 1 0 104 0 104 1.0000000 0.9542087 20 1 0 103 0 103 1.0000000 0.9542087 27 0 1 102 1 101 0.9901960 0.9448537 29 1 0 101 0 101 1.0000000 0.9448537 47 0 1 100 1 99 0.9900000 0.9354052 51 0 1 99 1 98 0.9898990 0.9259567 53 1 0 98 0 98 1.0000000 0.9259567 55 2 0 97 0 97 1.0000000 0.9259567 56 4 0 95 0 95 1.0000000 0.9259567 . . . . . .

t = time (days), c = number censored, d = number diedn = number at risk, s = number surviving to next time = n–dp = proportion surviving this time = s/nP = cumulative proportion surviving = p × previous P


We continue on down the table until all the days

have been used up. We omit days when there are

no events and no censorings, because they alter

neither the proportion surviving nor the number at

risk. The proportions estimated to survive from the

start to each time are the Kaplan Meier survival

estimates.

The Kaplan Meier survival curve

A table with tens or even hundreds of survival

estimates is pretty cumbersome and we usually

present the Kaplan Meier survival analysis

graphically. Figure 1 shows the Kaplan Meier

survival curve for the Control group.

Figure 1. Kaplan Meier survival curve for the Control group

We can compare the two arms of the trial, as

shown in Figure 2. We can see that Spironolactone

appears to have better survival, because the curve

is above that for the Control group.

Figure 2. Kaplan Meier survival curves for the Spironolactone and Control groups

The survival estimate is only an estimate, and if we

were to repeat the trial with a new sample of dogs

we would get a different survival curve. To allow

for this uncertainty, we can add a 95% confidence

interval for the survival estimate. This gives what

are called Greenwood bounds around the survival

curve. This gives a range within which we estimate

the survival for all dogs in the population to be.

Figure 3 shows these for the Control group.

Figure 3. Kaplan Meier survival curve for the Control group with Greenwood

confidence limits added

0.0

0.2

0.4

0.6

0.8

1.0

Proportion surviving

0 100 200 300 400 500Time (days)

Spironolactone Control

0.0

0.2

0.4

0.6

0.8

1.0ionsurviving

0 100 200 300 400 500Time (days)

0.0

0.2

0.4

0.6

0.8

1.0


0 100 200 300 400 500Time (days)

Control


The survival curve shows the estimated proportion

surviving at any chosen time, but we can also

estimate the time to which any chosen proportion

of dogs will survive. For example, we might want

to find the median survival time, the time to which

half the dogs survive. We draw a horizontal line

through the chosen proportion, 0.50, to intersect

the survival curve (Figure 4).

Figure 4. Attempt to estimate the median survival time for the Control group

However, the survival curve and the horizontal

line do not meet. We cannot estimate the median

survival time, because too few dogs have died at

the time of follow-up.

Figure 5. Estimation of the 75% survival time for the Control group

We can find the 75% survival time, when 25% of

the dogs have died, as shown in Figure 5. We draw

vertically down to the time axis to estimate the 75%

survival time as 335 days.

Figure 6. Confidence interval for the 75% survival time for the Control group

We can find the 95% confidence interval for this

estimate by drawing downwards from where our

75% line intersects the Greenwood bounds, as

shown in Figure 6.


But it is not a precise estimate. The lower limit of

the 95% confidence interval is 154 days. However,

the upper limit does not exist. The 0.75 survival line

does not cross the upper Greenwood bound. Too

few dogs have died for us to produce a meaningful

estimate.

The logrank test

We want to be able to compare the survival in

the treatment groups, to say whether there is

good evidence for a real difference between the

treatments (that is, whether the difference is

statistically significant) and to estimate how big

that difference is.

Greenwood standard errors and confidence

intervals for the survival probabilities are useful

for estimates such as the one year survival rate.

They are not a good method for comparing survival

curves. They do not include all the data and the

comparison would depend on the time chosen.

Instead of the Greenwood method, to compare

survival curves we need a method which makes

use of the full survival data. There are several

significance tests which we can use for this, of

which the best known is the logrank test. This is

a non-parametric test which makes use of the full

survival data without making any assumption about

the shape of the survival curve.

The logrank test tests the null hypothesis that, at

any time, the chance of a member of a group

experiencing an event is the same for both groups,

though the actual chance of an event may change

over time. The alternative hypothesis is that at

some time the chance of an event is different in the

two groups, which would make the survival curves

different.

We calculate how many of the 28 deaths observed

would be expected to be in the Spironolactone

group and how many would be expected in the

control group, if there were no difference between

Spironolactone and placebo. The precise number

expected depends on the pattern of censoring in

the groups. On each day where there are deaths,

for each group we calculate the total number of

deaths for the day divided by the number in the

group. Thus we split the number of deaths between

the two groups in proportion to the numbers at

risk. We then add these for each group. For the

trial data, we would expect 13.11 deaths in the

Spironolactone group and 14.89 deaths in the

Control group. We actually observed 6 deaths in

the Spironolactone group and 22 deaths in the

Control group. The probability of a difference this

large between what we observe and what we would

expect is 0.007. The difference is statistically

highly significant.

The hazard ratio

To produce an estimate of the size of the difference

in survival, we have to make some assumptions

about the shape of the curve. We have to assume

that they are similar in some way, so that we can

find some numerical value to compare between

them. We can use the Greenwood standard errors

to find a confidence interval for the difference

between the survival probabilities at a given time,

but this does not use all the data, events after the

chosen time being ignored.

The best way to estimate the difference between

the survival curves uses the hazard, which is

a measure of the chance that a member of the

population will have an event at any given time,

0.0

0.2

0.4

0.6

0.8

1.0


0 100 200 300 400 500Time (days)

Control group

335 days0.0

0.2

0.4

0.6

0.8

1.0


0 100 200 300 400 500Time (days)

Control group

335 days0.0

0.2

0.4

0.6

0.8

1.0


0 100 200 300 400 500Time (days)

Control group


or the rate at which events happen. To be more

precise, we find the probability of an event in any

small time interval by multiplying the width of the

time interval by the hazard at that time. Hazard

depends on the survival time, so that it might

increase or decrease as follow-up goes on. If we

can assume that the survival curves in the two

treatment groups follow the same pattern, then we

can assume that if hazard is greater in one group

than in the other at one time, it will also be greater

at another time. If this is the case, we assume

that the hazard in one group is equal to the hazard

in the other group multiplied by a constant number,

which we will estimate. Thus, if members of one

group have twice the risk of an event for members

of the other group on the first day, they will also

have twice the risk of the event on the second day,

twice the risk on the third day, and so on.

The constant ratio is called the hazard ratio. If the

risk of an event is the same in the two groups, the

hazard ratio is equal to one. If the risk is lower in

the intervention group than in the control group, the

hazard ratio is less than one. If the risk is greater in

the intervention group, the hazard ratio is greater

than one. For the hazard in the Spironolactone group

divided by the hazard in the Control group, the hazard

ratio is 0.31 with 95% confidence interval 0.13 to

0.76. At any given time, the risk of death in the

Spironolactone group is estimated to be one third of

that in the Control group.

We can adjust the treatment hazard ratio for

other variables which may influence survival,

using a method called Cox proportional hazards

regression. One obvious variable to adjust for is

study of origin, the dogs having originally been in

two different medium-term studies. These were

then combined to produce the long-term study.

The adjustment has no discernable effect,

the hazard ratio and confidence interval being

unchanged to two decimal places.

Other variables were used in the earlier analysis

as potential predictors of survival: the duration of

cardiac therapy before inclusion, and concomitant

treatment with furosemide. In Cox regression, we

usually limit the number of variables to at most

one per 10 events. We have 28 deaths among the

dogs, so here the maximum number of predictors

for a reliable analysis is three. We can add each of

these variables to the regression on treatment and

original study, separately. When we do this, there

is virtually no effect on survival and the hazard ratio

for treatment with Spironolactone remains almost

unchanged.

Presenting the results of survival analysis

The hazard ratio is the usual statistic used to

report the results of survival analysis in the

medical literature, where such analyses are much

more common than in the veterinary literature.

For example, we can see this in major medical

journals. The Lancet is one of the most highly cited

international medical journals and reports many

studies using survival data. In the first three months

of 2007, there were 13 papers which reported the

results of survival data in the Summary. Of these

13 papers, 12 quoted hazard ratios or statistics

derived directly from them, such as percentage

reduction in the mortality rate, 7 quoted Kaplan

Meier survival proportion estimates, and none

quoted median survival time estimates or any other

estimates of survival times.


Of these 13 papers, 10 reported randomised

trials. The main hazard ratios for these trials were:

0.40, 0.47, 0.64, 0.66, 0.69, 0.76, 0.78, 0.81,

0.83, 0.97. (Where hazard ratios were greater

than 1.0, I have reversed the order of treatments

to make them comparable to the Spironolactone

trial). All of these were closer to 1.0 (no effect)

than the 0.31 observed for Spironolactone and the

mean hazard ratio = 0.70. Hence this trial has

produced an unusually large effect.

Survival analysis has proved so valuable that Kaplan

and Meier (1958) and Cox (1972) are the two

mostly highly cited statistical papers to date (Ryan

and Woodall, 2005).

References

Cox DR. (1972) Regression models and life tables, Journal of the Royal Statistical Society, Series B 34, 187-220.

Kaplan EL. and Meier P. (1958) Nonparametric estimation from incomplete observations. Journal of the American Statistical Association, 53, 457-81.

Ryan TP and Woodall WH (2005) The most-cited statistical papers. Journal of Applied Statistics 32, 461-474.


AnsWers From:

clAUdio bUssAdori (cb)dVm, md, dipecVim, phd – director, clinica Veterina Gran sasso and researcher, paediatric cardiology department, s. donato Hospital, milan, italy

JoHn mArtin blAnd (Jmb)phd, msc, dic, Arcs, Fss, Hon. mrcr – professor of Health statistics, department of Health statistics, University of York, York, UK

lAUre bAdUel (lb)dVm, Head of the pre-clinical and clinical department, ceva santé Animale

Question (Delegate) - How can we keep confidence in trial, such as this, where there is a high censor rate?

Answer (JMB) - Many censorings were because owners did not wish the dog to continue with the

trial. The proportion of dogs which were withdrawn at the end of the short-term studies or which

died was very similar in the two groups. This is a double blind trial and neither owners nor vets knew

which treatment the dog was receiving, so they did not withdraw because they were not happy with

the treatment to which the dog had been allocated. Survival curves were drawn considering censored

data. The efficacy of spironolactone was demonstrated comparing these two curves.

Comment (Speaker: Faiez Zannad, France) - A mortality trial is not designed to identify how a

treatment works. One way of looking at whether spironolactone had a diuretic effect would be

to compare the doses of frusemide in the treatment and control groups. If there was a diuretic

effect of spironolactone, then one would expect a reduced dose of frusemide in the treated dogs.

Answer (LB) - We attempted to follow the frusemide doses and found no differences between the two

groups in mean dosages used. But we were limited in this assessment because of clinical treatment

reasons, for example whether the frusemide was given by intravenous or oral routes.

Question (Speaker: Nicolette Farman, France) - Is there a difference between the responses of male

and female dogs to spironolactone?

Answer (LB) - We observed no difference between sexes in response.

Answer (CB) - We must remember that it is male dogs that are more frequently affected by

myxomatous mitral valve disease.

Question (Speaker: Johann Bauersachs, Germany) - First, have you had a chance to look at the

echocardiographic data? Second, did you prevent the development of hypokalaemia at the dose of

spironolactone used?

Answer (CB) - We haven’t yet looked at the echocardiography data, largely for the reason that there

is too much variability in the methods used to measure different echocardiographic parameters by

clinicians across Europe. This is always a difficulty in this type of study. As for your second question,

hypokalaemia is rare in my experience and limited to a few cases that develop trembles and a loose

neck carriage. These are usually seen when the dose of furosemide is >5mg/kg twice daily. I haven’t

seen problems when combining ACE inhibitor and furosemide.

Question (Delegate)- The number of cases in the clinical trial were in ISACHC (International Small

Animal Cardiac Health Council) stage III (advanced heart failure) was very low. Should you be able

to exclude these and look specifically at those in stage II (mild to moderate heart failure)?

Answer (LB) - We haven’t done that; the trial wasn’t designed to do that.

Answer (JMB) - If the disease characteristics are important, then one can test for interaction but

the trouble is that these tests are not very powerful and you would need four-times as many dogs as

simply looking at treatment effects.

Question (Chair) - In veterinary medicine, where there is the option for euthanasia, how do you best

account for this end point? Especially given that different owners may differ in their preference

or decision making.

Answer (JMB) - The answer is simple. Do a double-blind, placebo-control study, then nobody knows

the treatment and cannot alter the decision to euthanase.

Question - Is there any validity in censoring these data?

Answer (JMB) - You should definitely not censor these data. You should only do that where deaths are

due to other causes, e.g. road traffic accidents.

Question (Delegate) - How do you prove the diuretic effect of spironolactone? Is there any evidence

of weight loss or oedema reduction?

Answer (CB) - There was no direct evidence of the diuretic effect, although we were not looking

specifically at this.

Answer (LB) - We sampled urine, but only collected a single sample every 1-3 months and this wasn`t

always at the same time each day. It is therefore very difficult to interpret the results and assess the

diuretic effect. It is not like in experimental conditions where you can collect urine over a 24 hour period.

Answer (CB) - In reality it is impossible to differentiate in this study between the effects of frusemide

and spironolactone on diuresis.

qUestion sessionclAUdio bUssAdori (cb), JoHn blAnd (Jb) And lAUre bAdUel (lb)


Question (Chair) - I would like to turn that back on Professor Hamlin and how much additional time

would make you confident to prescribe a drug?

Answer (Hamlin) - A small (2 mmHg) change in blood pressure brings about a 7% reduction in mortality

due to heart failure and a 10% reduction in stroke but this is the result of large metanalysis of millions

of patients. So small changes can make a big difference. I have no answer to your specific question.

Some clients want 2 days, others 6 months. While regulatory agencies make no specific limits, they

would not approve a drug unless it had a clinically meaningful effect.

Question (Speaker: Frédéric Jaisser, France) - Regarding median survival times, how long would we

have to wait to identify this value for the spironolactone treated dogs?

Answer (JMB) – If this had been a human trial, the results were such that it would have been ethically

essential that the trial was stopped and all patients offered the treatment. So, to answer your

question, we should not let dogs go on dying just to get that figure. They must be treated. It is

impossible to calculate the median survival time from the present data and we would have had to wait

several years to get to that point.

Answer (LB) – There is data from a study done by Michele Borgarelli showing that the median survival

time of dogs in Stage II ISACHC heart failure is 33 months.

Comment (Speaker: Faiez Zannad, France) - Regarding clinical meaningfulness and regulatory

agencies, the lowest outcome that I have seen in trials approved by these agencies is 8%. But

then how much would you, as a patient, be prepared to pay for such an outcome? So, there is no

single way to look at clinical meaningfulness. The regulatory agencies simply check that it is of

robust statistical significance.

Comment (Chair) - In my experience the FDA (Federal Drug Agency) only accepts clinically

significant evidence, and not biomarkers. For instance they won’t accept urinary protein as an

end-point – they want to see the clinical benefit.

Question (Chair) - Just to follow up Dr Schneider’s point, Claudio indicated that the dogs were

in mild to moderate Mitral Valve Disease. Did you exclude those with pulmonary oedema on

radiographs?

Answer (CB) - Yes, those with acute pulmonary oedema on thoracic radiographs were excluded.

Question (Chair) - Did any of the dogs have primary respiratory disease as the cause of their cough?

Answer (CB) - We did chest films in all cases in an effort to exclude these cases.

Answer (LB) - If we did miss them, then there is every likelihood that such cases would be balanced

between the spironolactone and control groups.

Question (Delegate) - I have a comment as to the potassium status. People, dogs and cats are all

different. It is amazing how tolerant dogs are to hypokalaemia.

Answer (CB) – I agree, dogs are very different in this respect.

Question (Speaker: Robert Hamlin, USA) - Does the EMEA (European Medicines Agency) draw a

distinction between clinically and statistically significant differences?

Answer (JMB) – I can’t respond on behalf of the EMEA but I would say that, just because something

is statistically significant that doesn’t make it clinically important. The evidence from this trial is that

spironolactone is associated with better survival, but how much better?

Question (Speaker: Robert Hamlin, USA) - So, what is clinically meaningful?

Comment (Chair) - John Rush and colleagues at Tufts did a study looking at what owners value in

terms of time and quality of life with their pets. The majority selected a period of 6 months but

would be happy with an additional 3-6 months.

Answer (CB) - It is difficult to understand what quality of life means. Owners accept very different

outcomes; it is a subjective matter and difficult to predict the views of individual owners.

Answer (JMB) - I would only add that when Richard Peto launched his call for large but simple clinical

trials, he was only looking for a 10% reduction in mortality as a measure of significance.


Faiez Zannad is Professor of Therapeutics and Cardiology. He is at the Head of the Division of Heart Failure, Hypertension and Preventive Cardiology for the department of Cardiovascular Disease of the Academic Hospital (CHU) in Nancy and the Director of the Clinical Investigation Centre (INSERM-CHU) of Nancy since 1995. He entered the European Society of Cardiology (ESC) in 1996 and is currently the Chairman of the ESC Working group on Pharmacology and Drug Therapy as well as a Board member of the ESC Heart Failure Association. He is Past-President of the French Society of Hypertension.

As the Coordinator of French Cardiovascular Clinical Investigation Centres, he has participated in various famous large scale trials in human cardiology such as RALES, VALIANT, CIBIS, CAPRICORN or EPHESUS. In these trials, he has been involved either as a member of the Steering Committees or in the Protocol Writing Groups.He is Co-Editor-in-Chief for Fundamental and Clinical Pharmacology, the official journal of the European Pharmacology Societies Federation (EUPHAR). He chairs and organises annual international meetings on CardioVascular Clinical Trials (CVCT) and on Biomarkers in Heart Failure.

cUrrent clinicAl stAtUs And FUtUre directions For biomArKers in HeArt FAilUre

Introduction:

Biomarkers provide the clinical cardiologist with

a number of laboratory tests for defining the

molecular diagnosis, assessing new risk factors,

and better targeting the pharmaceutical

approaches in patients with cardiovascular

disease. An increasing number of novel risk factors

have been added to the classical risk factors of

cardiovascular disease. The recent surge of genetic

analysis procedures will likely soon.

This review is focused on the current clinical

status and future directions for biomarkers in

heart failure. All major biomarkers currently used

in clinical practice, and other candidate biomarkers

are listed in Table 1, from an excellent overview 1.

Overview of Current Knowledge

Natriuretic Peptides

Three peptides comprise the natriuretic peptide

family, including atrial natriuretic peptide (ANP),

BNP, and C-type natriuretic peptide. Elevations

of these peptides have been associated with

varying degrees of cardiac impairment. Two forms

of natriuretic peptide, BNP (B-Type Natriuretic

Peptide) and its precursor, NT-Pro BNP, have

been studied as aids to establish the diagnosis,

estimate prognosis and monitor the response to

therapy of patients with ADHF 2. Recent studies

have established their diagnostic value in patients

presenting with dyspnœa, their association with

prognosis and early results suggest their potential

role in assessing response to therapy in ADHF.

Natriuretic peptides are attractive biomarkers in

HF for several reasons. An ideal biomarker should

possess a high degree of sensitivity and specificity

for the disease state which has been demonstrated

for natriuretic peptides in HF. Their enhanced

gene expression is evident in early HF, and plasma

levels increase as HF progresses 3. Natriuretic

peptides are closely linked to the pathophysiology

of HF, as their actions include diuretic, natriuretic,

vasodilatory, and anti-fibrotic effects, and they

suppress renin angiotensin aldosterone system

activity. The biologic responsiveness to these

physiologic actions is blunted in patients with HF.

Multiple hypotheses have been proposed to explain

this observed resistance to the biologic effects of

natriuretic peptides 4. Numerous clinical studies

described below point to their clinical utility in HF.

Diagnostic Utility

The diagnostic value of BNP was evaluated in the

Breathing Not Properly study 5. BNP levels were

significantly higher in patients with dyspnea due to

congestive heart failure. McCullough et al reported

the diagnostic accuracy for physician clinical

judgment, BNP, and both 6. The diagnostic accuracy

associated with BNP levels >100 pg/mL was 81%,

Faiez ZannadMD, PhD

Professor of Therapeutics and Cardiology

Director, Clinical Investigation Center INSERM Head, Heart Failure and Hypertension Unit

Department of Cardiology, CHU and University Henri Poincaré,

Nancy, France



compared with 74% for clinical judgment alone.

Combining BNP with clinical judgment resulted

in an area under the ROC curve of 0.93 6. In the

PRIDE study 7, NT-Pro BNP results were correlated

with a clinical diagnosis of acute HF. NT-Pro

BNP levels increase with age so that the study

investigators recommended cut points of

> 450 pg/ml for patients younger than 50 years

of age and > 900 pg/ml for patients age 50 years

or older. These cut points were associated with

highly sensitive and specific for HF in this study.

Prognostic Utility

Serial measurements of BNP appear to provide

prognostic information beyond that achieved with

a single baseline determination 8. Patients whose

BNP increased from baseline to 4 months had a

higher mortality risk as compared to patients

whose BNP stays below the median throughout the

study. Increasing BNP was also associated with

increases in left ventricular end-diastolic diameter,

whereas decreasing BNP was associated with

decreases in this measure 8. These data suggest

that monitoring BNP change during follow-up may

aid in patient risk stratification.

Challenges Facing Natriuretic Peptides

as Biomarkers

While natriuretic peptides are well validated as

biomarkers in HF, several cautions related to the

clinical use should be recognized. Consistency

in the method used to serially monitor BNP is

important. Assay results for BNP may vary widely

due to differences in antibody cross-reactivity 9, 10.

In addition, demographics and comorbidities such as

female gender, renal impairment, obesity, and atrial

fibrillation may significantly influence natriuretic

peptide values independent of the degree of HF

present. Whereas BNP is an established indicator

of decompensated HF, substantial variation in BNP

levels have been reported for ambulatory patients

with chronic heart failure 11. As discussed in detail

below, several recent reports question whether

assays used in clinical practice are actually

measuring the biologically active form of natriuretic

peptides 12-15. These findings indicate that altered

forms of BNP contribute to the values detected

by various clinical assays. Whether more specific

measurement the active form of BNP would be of

greater clinical value than existing assays, remains

to be determined. Other natriuretic peptide gene

products could be useful HF biomarkers, but

more research is needed to better identify them

and determine their diagnostic and prognostic

significance.

Necrosis Markers

Cardiac troponins are well established as the markers

of choice for myocardial injury and necrosis in acute

coronary syndromes. They appear in the plasma

rapidly after myocardial injury with a high sensitivity

and specificity 16. Cardiac troponins are detectable

even when creatine kinases are not and the troponin

T and I isoforms are unique for cardiac tissue. Recent

studies have also documented increased circulating

troponin in HF patients in the absence of acute

ischemia, leading to extensive investigation of this

molecule as a biomarker in this syndrome.

Association of Troponin with Clinical Outcome

Although elevations are modest compare to those

seen in acute coronary syndrome, several studies

have demonstrated an association between

abnormal troponin levels and subsequent clinical

events in HF 17-19. Serial measurements of

troponin may also have important value. Troponin


release during follow-up is associated with a

higher mortality risk and a higher risk of heart

failure hospitalization. Abnormal troponin release

is an attractive biomarker for HF because it seems

to reflect loss and/or progressive dysfunction of

cardiac myocytes. Troponin may be useful in the

selection of patients who may be appropriate

candidates for therapies targeting the prevention

of myocardial necrosis. Troponins may also be

valuable as components of a multimarker strategy

to identify high risk patients. Although troponin

release seems to reliably identify myocyte injury,

it does not indicate a specific mechanism of

injury. Further research should provide a better

understanding of the mechanism(s) of troponin

release and may lead to the identification of other

clinically important markers of the underlying

pathophysiological process of cardiomyocyte

compromise and loss. Elevated troponin is a

consistent predictor of mortality in HF. However,

additional studies are needed to determine

troponin suitability as surrogate marker of

response to drug therapy.

Fibrosis Markers 20-22

The extracellular cardiac matrix (ECCM) plays an

important role in the support of myocytes and

fibroblasts. Collagen is the principal structural

protein and collagen types I and III are the most

abundant in the myocardium. Collagen type I has

a poor specificity but represents the majority of

cardiac collagen (85%) and confers tensile strength

and resistance to stretch and deformation.

Type III is less abundant but more specific to the

heart and confers resilience 1-3. Fibrillar collagens

within the myocardium are substrates for matrix

metalloproteinases (MMPs). Among the MMPs,

MMP-1 has the highest affinity for fibrillar collagen

and preferentially degrades collagen I and III.

The net level of MMP-1 activity is dependent on

the relative concentrations of active enzyme and of

a family of tissue inhibitors of metalloproteinases

(TIMP). MMP-1 and TIMP-1 are co-expressed in

cardiac fibroblasts and are tightly regulated to

maintain the architecture of the ECCM. CITP is

a pyridinoline–cross-linked telopeptide produced

as a result of the hydrolysis of collagen type I

fibrils by MMP-1 and is a marker of collagen type

I degradation. The disruption of the equilibrium

between the synthesis and degradation of the

ECCM results in an excessive accumulation of

collagen type I and III fibers within the myocardium.

ECCM remodeling is an essential process in cardiac

remodeling, hypertensive cardiac hypertrophy,

dilated cardiomyopathy, and post infarction healing.

ECCM turnover is influenced by ischemia, stretch,

inflammation, and neurohormonal mediators.

Myocardial fibrosis is therefore the consequence

of a number of pathologic processes mediated by

mechanical, neurohormonal, and cytokine factors.

Cardiac fibrosis, a major determinant of diastolic

dysfunction and pumping capacity, results in

tissue heterogeneity and anisotropy, provides

the structural substrate for dys-synchrony and

arrhythmogenecity, thus potentially contributing

to the progression of congestive heart failure

(HF) and sudden cardiac death. ECCM turnover

may be the target of therapeutic agents aimed

at preventing or limiting the progression of

adverse cardiac remodeling in HF and therefore

hospitalization for HF as well as death due to

progressive HF and sudden cardiac death.

Given the importance of fibrous tissue in the

pathophysiology of myocardial dysfunction and

failure the non-invasive assessment of fibrosis

could prove to be a clinically useful tool, particularly

given the potential for cardioprotective and

cardioreparative pharmacological strategies.


The measurement of various serum peptides

arising from the metabolism of collagen types I

and III may provide information on the extent of

myocardial fibrosis and thus prognosis as well

as clues to appropriate strategies to improve

prognosis. Since procollagen type I C-terminal

propeptide (PICP), aminoterminal propeptides of

type-I procollagen (PINP), and N terminal type III

collagen peptide (PIIINP) are released with collagen

type I or III molecules in a stoichiometric manner

during collagen biosynthesis, they are important

markers of this process. Although these markers

are not specific to the myocardium, studies have

shown a correlation between myocardial collagen

content and the serum concentration of PICP in

patients with hypertension and have demonstrated

that serum PICP is secreted by the heart via the

coronary sinus in patients with hypertensive heart

disease. The PIP/CITP ratio, an index of coupling

between the synthesis and degradation of collagen

type I, was found to be higher in hypertensive

patients with increased collagen accumulation

in myocardial tissue than in those with normal

collagen accumulation. This evidence linking serum

ECCM markers to the heart ECCM content

provides a rationale for their use as biomarkers of

ECCM remodeling in cardiac disease. MMP-1 and

TIMP- 1 levels in coronary sinus blood are higher

than in peripheral venous blood in hypertensive

patients, but not in normotensive subjects,

although there was no association between blood

levels and myocardial expression of MMP-1 and

TIMP-1 or the amount and distribution of fibrillar

collagen.

Biomarkers reflecting collagen formation and/or

degradation may be used for early detection

of otherwise sub clinical disease; diagnostic

assessment of acute or chronic clinical syndromes;

risk stratification of patients with confirmed

disease; selection of appropriate therapeutic

interventions; and monitoring the response to

these interventions. ECCM biomarkers in patients

with HF may detect early changes in heart and

large vessel structure and function, the transition

to heart failure, and prognosis. The ability of

treatment to reduce myocardial fibrosis in patients

with HF may be monitored by the measurement of

various serum peptides arising from the metabolism

of collagen types. Characterization of patients

according to the severity of cardiac fibrosis, as

assessed by ECCM biomarkers, may prove useful

for selecting appropriate drug regimens, such as

aldosterone antagonists. The available data set

the stage for large scale long-term randomized

trials to validate this approach. However, before

wide spread application of this approach it will be

necessary, as pointed out above, to standardize the

various measurements of ECCM biomarkers and to

recognize their limitations. In part these limitations

may be overcome by the concomitant use of new

imaging techniques to localize myocardial fibrosis

such as radionuclide angiography with labeled

intergrins as well as magnetic resonance imaging.

Multimarker Strategy

Since several biomarkers appear informative for

the diagnosis and prognosis of HF, interest has

developed in the potential clinical use of multiple

markers simultaneously in individual patients.

Combining biomarkers incorporates potentially

additive information and may provide a more

accurate representation of a patient’s risk

profile. Ishii et al studied 98 patients hospitalized

for decompensated HF 23. Troponin T, troponin I,

and BNP were obtained at the time of admission.

The optimal value of troponin and BNP to predict


clinical events was determined by a receiver

operating curve approach. Patients were

categorized into low, intermediate, and high risk

groups according to troponin and BNP levels. As

expected, low risk patients had low troponin and

low BNP. Intermediate risk patients had either

troponin or BNP above the optimal prediction level,

while high risk patients had both troponin and

BNP above the optimal prediction level. The

combination of troponin and BNP provided a more

accurate risk stratification than either of the

parameters alone 23. The authors of this study

replicated their findings in a separate patient

cohort 24. Similar findings have also been reported

by Horwich et al. Patients with elevations of both

BNP and troponin had the highest mortality in a

cohort of 238 patients with advanced HF undergoing

transplant evaluation 19.

Multimarker strategies are attractive because

they allow the integration of several components

of the pathophysiologic process. Combining BNP

and troponin values incorporates an assessment

of both congestion and myocardial injury. HF is a

complex syndrome with multiple pathophysiologic

processes that occur simultaneously. Adopting

a multimarker strategy may yield more accurate

diagnostic and prognostic information since it

incorporates more than one aspect of the disease

process.

Novel Potential Biomarkers in Heart Failure

(Table 1)

Cytokines: Interleukin 18

Changes in the elaboration of many cytokines have

been identified in HF but no specific cytokine has

emerged as a clinically useful biomarker to date.

The search for novel cytokines as biomarkers

in HF continues. Abnormally elevated levels of

interleukin-18 (IL-18) have recently been detected

in a small study of patients with heart failure, and

ANP mRNA expression increased in IL-18 treated

myocytes 25. In addition, IL-18 levels have been

associated with symptom severity. In a study of 86

patients with NYHA Class II-IV heart failure, IL-18

levels were significantly higher among patients

with NYHA Class IV symptoms as compared to

NYHA Class II or III 26. Another study reported

higher levels of IL-18 in patients with HF as

compared to normal controls, and IL-18 was also

associated with NYHA Class. In contrast in this

patient population, IL-6 was not associated with

functional class, and there was no detectable

correlation between IL-6 and IL-18 27. White et

al recently reported the results of a study in

29 patients with worsening HF who received

72 hours of milrinone or dobutamine therapy.

IL-18 was significantly higher as compared to age

matched controls on admission while at 30 days,

a significant reduction in IL-18 and other markers

of inflammation was observed 28. These studies

suggest the potential for IL-18 as a biomarker in HF

but additional studies will be required to determine

if this cytokine will be of clinical value in predicting

outcome or monitoring therapy in HF.

Other Natriuretic Peptides

The family of natriuretic peptides consists

o f severa l members , but most c l i n i ca l

recommendations focus only on the assessment

of BNP and its precursor NT-Pro BNP 29, 30.

Currently, both are considered superior to

atrial natriuretic peptide (ANP) for diagnostic

and prognostic assessment of patients with

HF, even though ANP comprises most of the

natriuretic peptides in the circulation 31, 32.

The assessment of ANP is less reproducible, but

measurement of ANP’s precursor proANP may

overcome the problem of reproducibility, since it


is significantly more stable in the circulation than

the mature peptide. Assessment of other

natriuretic peptides may prove valuable. The

predictive value of mid-regional proANP, a novel

natriuretic peptide was assessed in a cohort

of 525 patients with stable HF 33. Mid-regional

proANP was at least as powerful as NT-ProBNP in

predicting mortality in these patients. Mid-regional

proANP was superior to NT-ProBNP in important

subgroups of patients in which NT-ProBNP failed

to predict survival. This was particularly evident in

patients with mild disease or the obese. Additional

studies in other patient populations with HF are

needed to fully validate this marker, but these

studies provide support for the need to monitor

multiple markers. Other natriuretic biomarkers

such as mid-regional pro-adrenomedullin may also

prove to be useful 34.

Novel Biomedomic Approaches

Recent advances in molecular biology methods

promise to transform the current approach to

detection and application of biomarkers in heart

failure. Specific examples of recent data from these

rapidly evolving fields (based on proteomic and

transcriptomic methods) illustrate their potential

to fundamentally alter our approach to biomarker

characterization of HF.

Potential for biomarkers to guide therapy

Conceptually, the use of biomarkers to guide

therapeutic decisions is attractive and already

supported by a variety of observational studies

and a few preliminary clinical trials. However, no

biomarker to date has been clearly established

for this indication in HF. Interest continues to

be driven by the lack of precision in early clinical

diagnosis and lack of reliable indicators to guide

the therapy in established HF. Many HF patients

remain at high risk despite advances in therapy.

Using classical clinical assessments has not

prevented significant treatment gaps in the

use of evidence-based medication which persist

despite strongly positive results from randomized

clinical trials. Observational prognostic studies

demonstrate convincing evidence that biomarker

measurements provide a more accurate

assessment of risk than clinical assessment

alone and suggest that knowledge of biomarkers

could help optimize therapy in HF.

As a foundation and rationale for larger outcomes

studies, preliminary findings are now available

from several recent pilot trials on biomarker

guided therapy in HF. These results include findings

from The Strategies for Tailoring Advanced Heart

Failure Regimens in the Outpatient Setting:

Brain Natriuretic Peptide Versus the Clinical

Congestion Score (STARBRITE) trial and STARS

(Treatment monitoring of systolic cardiac

insufficiency), which were designed to evaluate

whether a BNP guided approach to managing

fluid balance is more effective at preventing

death or hospitalization as compared to clinical

assessment alone in patients hospitalized with

acute decompensated HF. These studies provide

substantial support for large-scale outcomes

studies of the efficacy of monitoring therapy based

on BNP.

Troponin is another candidate for consideration

as a marker to guide management of patients with

heart failure. Although the mechanisms responsible

for troponin release are not well understood,

elevated troponin detected by current assays does

appear to reflect true cardiac myocyte loss and has

been strongly associated with adverse outcomes.

These findings suggest troponin could serve as a


guide/surrogate marker of therapeutic response

in HF patients. The proposed rational concept

assumes that a therapeutic regimen associated

with increased troponin may have a negative

effect on clinical outcomes, whereas treatments

associated with normalization of troponin or no

troponin release is likely to be associated with

favorable outcomes. Whether troponin release is

secondary to ischemia, remodeling, or elevated

filling pressures, targeting these conditions with

adjustments of various therapeutic interventions

may result in reduced myocyte loss and improved

outcomes.

Despite early favorable data, there are still several

important aspects of biomarker guided therapy

to carefully consider in testing this strategy:

1) benefit and risk associated with specific

treatment adjustments in response to changes

in biomarkers; 2) use of single versus multiple

markers to guide therapy; 3) extent of change

in biomarker(s) necessary to alter therapy; and

4) target HF patient population for guided

therapy.

Table 1: From Braunwald E. Biomarkers in heart failure. N Engl J Med. 2008 May

15;358(20):2148-59



References

1. Braunwald E. Biomarkers in heart failure. N Engl J Med. 2008 May 15;358(20):2148-59.

2. Maisel A, Mueller C, Adams K Jr, Anker SD, Aspromonte N, Cleland JG, Cohen-Solal A, Dahlstrom U, DeMaria A, Di Somma S, Filippatos GS, Fonarow GC,

Jourdain P, Komajda M, Liu PP, McDonagh T, McDonald K, Mebazaa A, Nieminen MS, Peacock WF, Tubaro M, Valle R, Vanderhyden M, Yancy CW, Zannad F,

Braunwald E. State of the art: using natriuretic peptide levels in clinical practice. Eur J Heart Fail. 2008 Sep;10(9):824-39.

3. Luchner A, Stevens TL, Borgeson DD, Redfield M, Wei CM, Porter JG, Burnett JC, Jr. Differential atrial and ventricular expression of myocardial BNP during

evolution of heart failure. Am J Physiol 1998; 274:H1684-H1689.

4. Clerico A, Recchia FA, Passino C, Emdin M. Cardiac endocrine function is an essential component of the homeostatic regulation network:

physiological and clinical implications. Am J Physiol Heart Circ Physiol 2006; 290:H17-H29.

5. Maisel AS, Krishnaswamy P, Nowak RM, McCord J, Hollander JE, Duc P, Omland T, Storrow AB, Abraham WT, Wu AH, Clopton P, Steg PG, Westheim A,

Knudsen CW, Perez A, Kazanegra R, Herrmann HC, McCullough PA. Rapid measurement of B-type natriuretic peptide in the emergency diagnosis of heart failure.

N Engl J Med 2002; 347:161-167.

6. McCullough PA, Nowak RM, McCord J, Hollander JE, Herrmann HC, Steg PG, Duc P, Westheim A, Omland T, Knudsen CW, Storrow AB, Abraham WT, Lamba S,

Wu AH, Perez A, Clopton P, Krishnaswamy P, Kazanegra R, Maisel AS. B-type natriuretic peptide and clinical judgment in emergency diagnosis of heart failure:

analysis from Breathing Not Properly (BNP) Multinational Study. Circulation 2002; 106:416-422

7. Januzzi JL, Jr., Camargo CA, Anwaruddin S, Baggish AL, Chen AA, Krauser DG, Tung R, Cameron R, Nagurney JT, Chae CU, Lloyd-Jones DM, Brown DF,

Foran-Melanson S, Sluss PM, Lee-Lewandrowski E, Lewandrowski KB. The N-terminal Pro-BNP investigation of dyspnea in the emergency department (PRIDE) study.

Am J Cardiol 2005; 95:948-954.

8. Latini R, Masson S, Wong M, Barlera S, Carretta E, Staszewsky L, Vago T, Maggioni AP, Anand IS, Tan LB, Tognoni G, Cohn JN.

Incremental prognostic value of changes in B-type natriuretic peptide in heart failure. Am J Med 2006; 119:70-30.

9. Jortani SA, Prabhu SD, Valdes R, Jr. Strategies for developing biomarkers of heart failure. Clin Chem 2004; 50:265-278.

10. Vogeser M, Jacob K. B-type natriuretic peptide (BNP)--validation of an immediate response assay. Clin Lab 2001; 47:29-33.

11. Tang WH, Girod JP, Lee MJ, Starling RC, Young JB, Van LF, Francis GS. Plasma B-type natriuretic peptide levels in ambulatory patients with established chronic

symptomatic systolic heart failure. Circulation 2003; 108:2964-2966.

12. Hawkridge AM, Heublein DM, Bergen HR, III, Cataliotti A, Burnett JC, Jr., Muddiman DC. Quantitative mass spectral evidence for the absence of circulating brain

natriuretic peptide (BNP-32) in severe human heart failure. Proc Natl Acad Sci U S A 2005; 102:17442-17447.

13. Hino J, Tateyama H, Minamino N, Kangawa K, Matsuo H. Isolation and identification of human brain natriuretic peptides in cardiac atrium.

Biochem Biophys Res Commun 1990; 167:693-700.

14. Yandle TG, Richards AM, Gilbert A, Fisher S, Holmes S, Espiner EA. Assay of brain natriuretic peptide (BNP) in human plasma:

evidence for high molecular weight BNP as a major plasma component in heart failure. J Clin Endocrinol Metab 1993; 76:832-838.

15. Shimizu H, Masuta K, Asada H, Sugita K, Sairenji T. Characterization of molecular forms of probrain natriuretic peptide in human plasma.

Clin Chim Acta 2003; 334:233-239.

16. French JK, White HD. Clinical implications of the new definition of myocardial infarction. Heart 2004; 90:99-106.

17. La VL, Mezzena G, Zanolla L, Paccanaro M, Varotto L, Bonanno C, Ometto R. Cardiac troponin I as diagnostic and prognostic marker in severe heart failure.

J Heart Lung Transplant 2000; 19:644-652

18. Setsuta K, Seino Y, Takahashi N, Ogawa T, Sasaki K, Harada A, Takano T, Kishida H, Hayakawa H. Clinical significance of elevated levels of cardiac troponin T in

patients with chronic heart failure. Am J Cardiol 1999; 84:608-11, A9.

19. Horwich TB, Patel J, MacLellan WR, Fonarow GC. Cardiac troponin I is associated with impaired hemodynamics, progressive left ventricular dysfunction,

and increased mortality rates in advanced heart failure. Circulation 2003; 108:833-83

20. Zannad F, Rossignol P, Iraqi W. Extracellular matrix fibrotic markers in heart failure. Heart Fail Rev. 2009 Apr 29. [Epub ahead of print]

21. Iraqi W, Rossignol P, Angioi M, Fay R, Nuée J, Ketelslegers JM, Vincent J, Pitt B, Zannad F. Extracellular cardiac matrix biomarkers in patients with acute myocardial

infarction complicated by left ventricular dysfunction and heart failure: insights from the Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and

Survival Study (EPHESUS) study. Circulation. 2009 May 12;119(18):2471-9.

22. Radauceanu A, Ducki C, Virion JM, Rossignol P, Mallat Z, McMurray J, Van Veldhuisen DJ, Tavazzi L, Mann DL, Capiaumont-Vin J, Li M, Hanriot D,

Zannad F. Extracellular matrix turnover and inflammatory markers independently predict functional status and outcome in chronic heart failure. J Card Fail.

2008 Aug;14(6):467-74.

23. Ishii J, Nomura M, Nakamura Y, Naruse H, Mori Y, Ishikawa T, Ando T, Kurokawa H, Kondo T, Nagamura Y, Ezaki K, Hishida H. Risk stratification using a combination

of cardiac troponin T and brain natriuretic peptide in patients hospitalized for worsening chronic heart failure. Am J Cardiol 2002; 89:691-695.

24. Ishii J, Cui W, Kitagawa F, Kuno T, Nakamura Y, Naruse H, Mori Y, Ishikawa T, Nagamura Y, Kondo T, Oshima H, Nomura M, Ezaki K, Hishida H.

Prognostic value of combination of cardiac troponin T and B-type natriuretic peptide after initiation of treatment in patients with chronic heart failure.

Clin Chem 2003; 49:2020-2026.

25. Seta Y, Kanda T, Tanaka T, Arai M, Sekiguchi K, Yokoyama T, Kurimoto M, Tamura J, Kurabayashi M. Interleukin-18 in patients with congestive heart failure:

induction of atrial natriuretic peptide gene expression. Res Commun Mol Pathol Pharmacol 2000; 108:87-95.

26. Yamaoka-Tojo M, Tojo T, Inomata T, Machida Y, Osada K, Izumi T. Circulating levels of interleukin 18 reflect etiologies of heart failure:

Th1/Th2 cytokine imbalance exaggerates the pathophysiology of advanced heart failure. J Card Fail 2002; 8:21-27.

27. Naito Y, Tsujino T, Fujioka Y, Ohyanagi M, Okamura H, Iwasaki T. Increased circulating interleukin-18 in patients with congestive heart failure.

Heart 2002; 88:296-297.

28. White M, Ducharme A, Ibrahim R, Whittom L, Lavoie J, Guertin MC, Racine N, He Y, Yao G, Rouleau JL, Schiffrin EL, Touyz RM. Increased systemic inflammation and

oxidative stress in patients with worsening congestive heart failure: improvement after short-term inotropic support. Clin Sci (Lond) 2006; 110:483-489.

29. Adams KF, Lindenfeld J, Arnold J, Baker DW, Barnard D, Baughman KL, Boehmer JP, Deedwania PC, Dunbar SB, Elkayam U, Gheorghiade M, Howlett J,

Konstam MA, Kronenberg M, Massie B, Mehra M, Miller AB, Moser D, Patterson JH, Rodeheffer RJ, Sackner-Bernstein JD, Silver MA, Starling RC, III,

Stevenson LW, Wagoner L. Executive summary: HFSA 2006 Comprehensive Heart Failure Practice Guideline. J Card Fail 2006; 12:10-38.

30. Swedberg K, Cleland J, Dargie H, Drexler H, Follath F, Komajda M, Tavazzi L, Smiseth OA, Gavazzi A, Haverich A, Hoes A, Jaarsma T, Korewicki J, Levy S, Linde C,

Lopez-Sendon JL, Nieminen MS, Pierard L, Remme WJ. Guidelines for the diagnosis and treatment of chronic heart failure: executive summary (update 2005):

The Task Force for the Diagnosis and Treatment of Chronic Heart Failure of the European Society of Cardiology. Eur Heart J 2005; 26:1115-1140.

31. Vesely DL. Atrial natriuretic peptide prohormone gene expression: hormones and diseases that upregulate its expression. IUBMB Life 2002; 53:153-159.

32. Yoshibayashi M, Saito Y, Nakao K. Brain natriuretic peptide versus atrial natriuretic peptide--physiological and pathophysiological significance in children and adults:

a review. Eur J Endocrinol 1996; 135:265-268.

33. von Haehling S, Jankowska E, Morgenthaler N. Mid-regional pro-atrial natriuretic peptide (MR-pro ANP) as a novel and more stable prognostic marker in chronic

heart failure (CHF) [abstract]. J Am Coll Cardiol 2006; 47 (Suppl A):65A.

34. von Haehling S, Jankowska E, Morgenthaler N. Mid-regional pro-adrenomedullin as a novel and prognostic marker in chronic heart failure [abstract].

Circulation 2005; 112 (Suppl):II-670.


qUestion sessionFAieZ ZAnnAd

AnsWers From:

FAieZ ZAnnAdmd, phd – professor of therapeutics and cardiology, department of cardiology, cHU and University Henri poincaré, nancy, France

Question (Delegate) - First, what is the physiological basis for matrix metalloproteinases (MMP)

as cardiac biomarkers? Second, in which diseases have MMP been studied. Third, would you see

changes in MMP before end stage heart failure?

Answer - The questions relate to what extent the markers reflect collagen synthesis versus

degradation, and whether it is an acute or chronic disease state. MMPs are involved in collagen

degradation and are much more complex to measure than the other collagen biomarkers, suffer from

high variability and are unstable to store. This has limited their use.

Question (Delegate) - In order to circumvent daily variation within BNP and other biomarkers, are

there systems such as fructosamine that look at longer term values.

Answer - Biomarker companies are actively looking for more stable fragment, so-called memory

markers but these have not yet reached the clinical setting.

Question (Chair) - Would that include tests for proBNP?

Answer - It is a matter of where you are, since there are regional preferences for the way in which

tests are used.

Question (Chair) - Are there actually assays for the prohormone?

Answer - Yes, but it is unclear whether they measure the prohormone or specific fragments, and we

await more data.

Question (Chair) - If and how much does inter-daily and inter-weekly individual variability affect

cardiac biomarkers?

Answer - Synthesis biomarkers, like PIIINP, are extremely robust. BNP, however, is much more variable

from one day to another.

Question (Chair) - Do you know if a patients water intake or the amount of salt they had in their

diet or time of day influence what their value might be when they come into the hospital?

Answer - BNP has a very short turnover so it is very much related to congestion and hemodynamic

conditions. Collagen biomarkers are more stable, just because the turnover and the half-life are

extremely long. 120 days is the half-life of collagen renewal in the heart. There are short-term

variations in acute myocardial infarction or acute heart failure, for example, but in chronic conditions,

it is rather stable.

Question (Delegate) - There is an elevation of BNP in late heart failure. Is this due to resistance

to the peptide?

Answer - Ageing is important. BNP is very useful in acute heart failure trials as it indicates congestion.

But there is also a kind of tachyphylaxis in those patients with heart failure because they have high

level of BNP and so they are more resistant to BNP infusions.


Mark Oyama is an Associate Professor in the Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania. His main clinical and research interests involve canine myocardial disease, mitral valve disease and cardiac biomarkers. He is currently involved in several research projects dealing with investigation of biomarkers in dogs and cats as well as studies investigating serotonin-related molecular mechanisms of canine valve disease.

He is the current President of the American College of Veterinary Internal Medicine, Specialty of Cardiology and a member of the ACVIM Board of Regents. He has served as a member of the NIH-Center for Scientific Review Bioengineering, Technology, and Surgical Sciences study section and is a member of the University of Pennsylvania Institute of Translational Medicine and Therapeutics. Dr. Oyama has published over 90 scientific manuscripts and abstracts and has given over 150 national and international lectures. He serves on veterinary advisory boards for a variety of biotechnology, pharmaceutical, and diagnostic laboratory companies and is the Translational Sciences section editor for the Journal of Veterinary Cardiology. He resides in Philadelphia with a Golden retriever and two very mischievous cats.

Jonathan elliott MA VetMB PhD CertSAC DipECVPT MRCVS

Vice Principal – ResearchProfessor of Veterinary Clinical Pharmacology

Royal Veterinary CollegeLondon, England


mark A. oyama DVM, DipACVIM (Cardiology)

Associate Professor of CardiologyDepartment of Clinical Studies, School of Veterinary Medicine,

University of PennsylvaniaPhiladelphia, USA


Jonathan Elliott graduated from Cambridge Veterinary School in 1985. After a year as an Intern at the University of Pennsylvania, he undertook a PhD in vascular pharmacology in Cambridge. In 1990 he was appointed to a lectureship in Veterinary Pharmacology at the Royal Veterinary College and developed research interests in feline kidney disease and hypertension and Equine Laminitis. He was awarded the Pfizer Academic Award in 1998 and the BSAVA Amoroso Award in 2001, the Petplan Scientific Award in 2005 and the ESVNU Award in 2007 for contributions to companion animal medicine. He was a member of the ACVIM Consensus Statement Panels on Proteinuria and Hypertension and chaired the International Renal Interest Society from 2002-2004.

He is currently Professor in Veterinary Clinical Pharmacology and Vice Principal for Research at the RVC and is a Diplomate of the European College of Pharmacology and Toxicology and a member of the UK Government’s Veterinary Products Committee.

CHAIR ATTENDEES

LECTURES FROM P4 TO P27

Chairman

Chairman

LECTURES FROM P28 TO P63

AGUDELO Carlos, Czech Republic

BESSMANN Kai, Germany

BOSWOOD Adrian, United Kingdom

CADORE Jean-Luc, France

CHETBOUL Valérie, France

COLLINS Stephen, United Kingdom

D’AGNOLO Gino, Italy

DEINERT Michael, Germany

DEPREST Christine, Belgium

DIQUELOU Armelle, France

DUKES MAC-EWAN Jo, United Kingdom

FABRIES Lionel, France

FERASIN Luca, United Kingdom

GERRITSEN Rob, Netherlands

HAELEWYN Christine, France

HÄGGSTRÖM Jens, Sweden

HEZZEL Melanie, United Kingdom

HÖGLUND Katja, Sweden

JAMES Rachel, United Kingdom

KRESKEN Jan-Gerd, Germany

LEFEBVRE Hervé, France

LJUNGVALL Ingrid, Sweden

LOMBARD Christophe, Switzerland

MARKOVIC Mato, Germany

MARTIN Mike, United Kingdom

MARTINEZ-PEREIRA Yolanda, United Kingdom

MOGLIORINI Francesco, Italy

PASLAWSKA Urszula, Poland

PATTESON Mark, United Kingdom

PORCIELLO Francesco, Italy

POUCHELON Jean-Louis, France

ROURA Xavier, Spain

SANTAGIO José Antonio, Spain

SANTAMARINA German, Spain

SANTILLI Roberto, Italy

SCHNEIDER Matthias, Germany

SKRODZKI Marianne, Germany

SMITH Sarah, United Kingdom

SUMMERFIELD Nuala, United Kingdom

SWIFT Simon, United Kingdom

SZATMARI Victor, Netherlands

VAN ISRAEL Nicole, Belgium

VÖRÖS Károly, Hungary

WOTTON Paul, United Kingdom

Who are we?CEVA Santé Animale is the worlds 9th largest animal health company; we research, develop, produce and market pharmaceutical products and vaccines for companion animals, livestock and poultry. The company is based in Libourne, France and is directly invested in 45 other countries with distributive partners in many more, giving us a truly global dimension.

A results culture:

• Originally a subsidiary of the Sanofi group, the Sanofi Santé Nutrition Animale (SSNA) management conducted the first of three leveraged buy outs to launch in 1999 CEVA Santé Animale.

• In the latest LMBO completed during 2007, the CEVA Santé Animale’s management and employees assumed a majority stake in the company with the backing of financial partners Euromezzanine and Natixis.

During our first 10 years of existence, the company’s organic growth has averaged 8.4%, well beyond the industry average. We owe our existence to the farmers, pet owners and veterinarians who support us and as a result we are totally dedicated to providing them with products and services, that protect and improve not only the lives of their animals but the well being of us all, as a global community.

Key figures (Dec 2008):

Sales: Euro 363m.

Operating income: 49.4m.

R&D expenditure: 27.6m (7.7% sales)

Employees: 2175

30

25

20

15

10

5

02006

23.1

R&D Sales** Animal health without Toll-manufacturing and nutrition

2007

25.3

7.9% 7.8%

2008

27.6

7.7%

research & development ( M)

Geographic and strategic focus: GlocAlCeva Sante Animale is organised around 3 geographic zones, which correspond to the major regulatory and market-based blocks: Europe, North America and Zone International (Africa/Middle East, Asia/Pacific and Latin America).

Our objective is to direct resources towards developing products and services that correspond to the ever increasing specific requirements of local markets.

In turn the corporate marketing groups, working from head office, have global strategic responsibility across 3 target animal groups: companion animals, livestock and poultry.

Each group focuses it’s activity on the management of strategic products, further defined according to our areas of expertise:

Companion Animal: Behaviour, Cardiology, Locomotion.Livestock: Anti-invectives, Fertility management, Vaccines.Poultry: Pharmaceutical products, Vaccines and Equipment

Innovation is a frequently used word in the pharmaceutical world often associated with the introduction of new “blockbuster” products. Ceva’s approach to innovation, aside from our R&D programme, is to encourage our local and central teams to find better solutions to existing and emerging diseases, which will in turn improve animal health and productivity. That’ s what we mean when we use the word GLOCAL: our aim is to be global in our ambition to tackle the big issues and local in the way that we tailor practical solutions for our customers.

the bigger picture: 75% of emerging human diseases are of animal origin (source: oie)

Ceva is heavily involved in the fight to protect against diseases such as avian influenza and brucellosis that also directly menace the human population. Our strength and commitment in addressing these diseases goes far beyond our relative market position, this is equally true of our commitment to the emerging markets of the world, where we have invested significantly despite the level of risk.

We believe that a “one health, one planet” approach is required to properly address global animal health issues and we will continue to invest our time and resource to ensure that we make real impact.

North America 2 CEVA offices 2 R&D centres 2 Manufacturing sites 176 collaborators International Zone24 CEVA offices 1 R&D centres 7 Manufacturing sites 654 collaborators Europe20 CEVA offices 3 R&D centres 5 Manufacturing sites 1345 collaborators

2175 collaborators R&D Investments 7.7% fo 2008 TO

TO g 363 M

Prolong Life!*

*in combination with conventional therapy.

CEVA Santé Animale - www.ceva.comLa Ballastière - BP.126 - 33501 Libourne Cedex

D E V E L O P E D B Y C E V A S A N T É A N I M A L E

SPIRONOLACTONE

Aldosterone - Cardio Symposium 2009 · Aldosterone (Aldo) is the main regulator of sodium...

Documents

Transcript of Aldosterone - Cardio Symposium 2009 · Aldosterone (Aldo) is the main regulator of sodium...