Evidence for altered ETB receptor characteristics during

development and progression of ventricular cardiomyocyte

hypertrophy

Graham R. Lee, David Bell*, Elizabeth J. Kelso, Cymone C.H. Argent,

Barbara J. McDermott;

Department of Therapeutics and Pharmacology, Centre for Cardiovascular and

Genetics Research, School of Medicine, Queen’s University Belfast, UK.

Short Title: ETB receptor and cardiomyocyte hypertrophy in SHR

Corresponding author:

Dr. David Bell Department of Therapeutics & Pharmacology, The Queen’s University of Belfast, Whitla Medical Building, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, United Kingdom

Tel. +44 2890 335770 Fax: +44 2890 438346 Email d.bell@qub.ac.uk

Sources of Support: British Heart Foundation: Project grant PG1999150 PhD studentship FS98012

Articles in PresS. Am J Physiol Heart Circ Physiol (February 26, 2004). 10.1152/ajpheart.00461.2003

Copyright (c) 2004 by the American Physiological Society.

2

ABSTRACT

The hypothesis that endothelin (ET) receptor mechanisms are altered during

development and progression of left ventricular hypertrophy (LVH) in vivo was tested

using spontaneously hypertensive rats (SHRs). Ventricular cardiomyocytes were

isolated from SHRs prior to onset (8 and 12 weeks), and during progression (16, 20, 24

weeks) of LVH, and compared to age-matched normotensive Wistar Kyoto (WKY) rats.

PreproET-1 mRNA expression was elevated in SHR (P<0.05) relative to WKY

cardiomyocytes at 20-24 weeks. ET binding site density was 2 fold greater in SHR than

WKY cells at 12 weeks (P<0.05) but normalised at 20 weeks. ETB receptors were

detected on SHR cardiomyocytes as early as 8 weeks and their affinity increased

progressively with age (P<0.05), whereas ETB receptors were not detected on WKY

cells until 20 weeks. ET-1 stimulated protein synthesis with similar maximum

responses between strains (21-30%), in contrast to sarafotoxin 6c which stimulated

protein synthesis in SHR (13-20%) but not WKY cells at 12-20 weeks. In SHR, but not

WKY cells, the ETB receptor-selective ligand, A-192621, increased protein synthesis

progressively with the development of LVH (15% maximum effect). In conclusion, the

presence of ETB receptors (8-12 weeks), coupled with functional responsiveness of

SHR cells, but not of WKY cells, to sarafotoxin 6c at 12 weeks supports the

involvement of ETB receptors prior to the onset of cardiomyocyte hypertrophy, while

altered ETB receptor characteristics during active hypertrophy (16-24 weeks) indicate

that ETB receptor-mechanisms may also contribute to disease progression.

Key Words: left ventricular hypertrophy; pressure-overload; endothelin receptor;

endothelin-1

3

INTRODUCTION

Concentric left ventricular hypertrophy (LVH) occurs following pressure-overload and is

associated with thickening of the ventricular wall to normalise wall stress, but ultimately

leads to mechanical dysfunction and failure (24). Increased cardiac mass is attributed to

increased mass of individual cardiomyocytes, proliferation of non-myocytes and

synthesis of extracellular matrix (36, 40). Epidemiological studies have indicated that

regression of LVH with anti-hypertensive agents improves prognosis (27). Such

treatments, however, only partially regress LVH; involvement of non-haemodynamic

factors has also been postulated (9).

ET-1 is a potent vasoconstrictor peptide; ET-2 and ET-3 differ from ET-1 by 2 and 6

amino acids, respectively (44). Increased plasma levels of ET-1 occurs in hypertension

and heart failure and correlate with severity of LVH (15). ET receptor antagonists

attenuate LVH in some experimental models in vivo (16, 20). It is unclear if this occurs

as a direct result of blockade of ET receptors on cardiomyocytes, or represents an

indirect effect, due to reduction in systolic pressure; elevated ET-like immunoreactivity

and binding site density in cardiac tissue indicate that locally-derived ET may contribute

to ventricular remodelling (3, 35, 45, 6). The actions of ET are mediated by ETA and

ETB receptors, which are both present in the heart (39, 13): ETA receptors have greater

affinity for ET-1 than ET-3, while ETB receptors bind these peptides with equal affinity

(2). Sarafotoxin 6c (S6c) is an ETB receptor-selective agonist; BQ123 and ABT-627 (2-

(4-methoxyphenyl)-4-(1,3-benzodioxol-5-yl)-1-(N,N-di(n-butyl)amino carbonylmethyl)-

pyrrolidine-3-carboxylic acid) are selective antagonists at ETA receptors, while A-

192621 (2-(4-propoxyphenyl)-4-(1,3-benzodioxol-5-yl)-1-(2,5-ethylphenyl)amino

carbonylmethyl)-pyrrolidine-3-carboxylic acid) is a very potent and highly selective

antagonist at ETB receptors (16, 8). ET-1 elicits a positive inotropic effect on the

4

myocardium; both ETA and ETB receptors are implicated in the contractile responses to

ET-1 in normal and diseased cardiomyocytes (22).

ET-1, via ETA and ETB receptors, initiates increased mass of adult cardiomyocytes in

vitro, in which the influence of mechanical loading is eliminated (8). ETB receptor mRNA

is up-regulated in hypertrophying neonatal cardiomyocytes (19). ET-3 and mechanical

stretch induce expression of preproET-1 mRNA, while hypertrophy of neonatal

cardiomyocytes by each stimulus in vitro is attenuated by BQ123 (38, 43). Non-

myocytes provide an additional source of ET-1 and ET-3 within myocardium (41). ET

peptides may initiate cardiomyocyte hypertrophy with additional factors taking over a

maintenance role, since the initial attenuation by BQ123 of the onset of LVH following

aortic banding of adult rats is not sustained (16). These data highlight the importance of

longitudinal studies, utilising cells obtained ex vivo from diseased hearts, to address the

temporal dependence of expression by cardiomyocytes of preproET-1 and ET receptor

mRNA and the relative abundance of, and responsiveness to each receptor subtype

during onset and progression of LVH in vivo.

The spontaneously hypertensive strain of Wistar rat (SHR) provides a useful model of

human hypertension and LVH (11, 31). Hypertension develops gradually in SHRs a few

weeks after birth; onset of LVH occurs between 10-20 weeks. Despite severe

elevations of systemic arterial pressure, cardiac output is maintained initially by

moderate LVH. Since alterations in cardiac performance may reflect many influences

(intrinsic muscle properties, loading conditions, altered systemic and/or coronary

haemodynamics), studies in cardiomyocytes specifically are useful to dissect out

adaptations intrinsic to them from those of fibrosis and non-myocyte proliferation. We

have characterised the SHR comprehensively at cardiomyocyte level allowing precise

5

application of this model in investigations of pathogenetic mechanisms; hypertension is

followed by active hypertrophic growth between 16-20 weeks, evidenced by increased

cell mass and width, which subsequently decelerates at 24 weeks as stable

compensation is attained (4). There are conflicting data regarding whether plasma ET-1

levels are elevated in SHRs (23, 39). Chronic intervention with ET receptor antagonists

attenuates hypertension only when over-expression of ET-1 in blood vessel walls is

demonstrable (28, 29). Evidence that bosentan causes some regression of LVH without

an appreciable reduction in blood pressure indicates that ET-1 may exert a local

influence on cardiomyocyte hypertrophy independent of systemic pressor effects (20).

For this reason, it is important to examine expression of the peptide and alterations in

ET receptors and/or responsiveness within SHR hearts: conflicting evidence has been

obtained in this regard depending on the approach used, tissue source and sampling

time (5, 18, 32), and little evidence exists of effects in isolated cardiomyocytes (10).

The aim of this longitudinal study was to investigate whether alterations in the ET

receptors were initiated in cardiomyocytes prior to the onset of LVH in SHRs, and if so,

whether these alterations were associated with the development and progression of

ventricular cell hypertrophy. Appropriate comparisons were made using cardiomyocytes

isolated from age-matched normotensive WKY rats.

6

METHODS

Experimental model. Male WKY and SHRs were obtained from Harlan (UK) at 4 weeks

and maintained until sampling at 8, 12, 16, 20, 24 weeks of age. The study was

performed in accordance with Home Office Guidance on the operation of the Animals

(Scientific Procedures) Act 1986, published by Her Majesty’s Stationary Office, London.

Following deep anaesthesia of rats using isoflurane (Abbott Laboratories, UK), the

hearts were rapidly excised and placed in ice-cold saline, and blood collected from the

chest cavity into ice-cold tubes containing EDTA (2 mmol/L) and aprotinin (500 IU/L)

(Sigma Chemical Company, UK). Immunoreactive (ir) ET was extracted from plasma

using C18 Sep-Pak cartridges (Waters Associates, USA) and measured by RIA

(Phoenix Pharmaceuticals Inc., USA) (18).

Cardiomyocyte isolation. Excised hearts were cannulated through the ascending aorta,

and ventricular cardiomyocytes isolated by enzymatic digestion (collagenase,

0.4mg/ml) using Langendorff perfusion (8). After purification, cells were suspended at

1.5x105 viable cardiomyocytes/ml in a ‘creatinine-carnitine-taurine’ (CCT) medium

which consisted of modified glutamine-free medium M-199 supplemented with Earle’s

salts (Gibco, UK), HEPES (15 mM), creatinine (5 mM), l-carnitine (2 mM), taurine (5

mM), ascorbic acid (100 µM), penicillin (100 IU/ml) and streptomycin (100 µg/ml). The

medium was also supplemented with cytosine β-D arabinofuranoside (10 µM) to

prevent growth of non-myocytes (Sigma Chemical Company, UK) (8).

Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Total cellular RNA was

isolated by a modification of the acid guanidinium thiocyanate-phenol-chloroform

method of Chomczynski and Sacchi (7). First strand cDNA was synthesised from 2 µg

total RNA by reverse transcriptase (Reverse-iT kit, Abgene, UK). Gene specific primers

7

were based upon those previously reported (37). After initial denaturation at 94oC for 4

min, cycling profiles included specific annealing temperatures and cycle numbers

(ppET-1: 54oC, 31; ECE: 60oC, 28; ETA/ETB receptors: 55oC, 32; respectively), followed

by extension at 72oC for 60 s. PCR products were electrophoresed on 2% agarose gels

and stained with ethidium bromide. Gels were visualised under ultraviolet illumination

and analysed using a Gene Genius Gel documentation system with Gene Tools

analysis software (Syngene, UK). Band intensity was expressed as target mRNA to

GAPDH mRNA ratio.

Preparation of sarcolemmae. Viable cardiomyocytes were suspended in a HEPES (20

mmol/L) buffer containing protease inhibitors (Sigma Chemical Company, UK):

aprotinin (0.8 µmol/L), bacitracin (0.1 mmol/L), benzamidizine (0.1 mmol/L), EDTA (5

mmol/L), leupeptin (2 µmol/L), PMSF (0.1 mmol/L] and homogenised at 9500 r.p.m.

(Ultra-Turax-T25, Janke+Kunkel, Germany) for 30 s. Disrupted cells were centrifuged

(2000 r.p.m., 5 min, 4oC) (Mistral MSE 400, UK) to sediment cell nuclei and

mitochondrial fractions, and supernatants then centrifuged x3 at 20000 r.p.m. for 30

min, 4oC, and the pellets stored at -70oC.

Homologous/heterologous competition binding. Sarcolemmae were suspended (20

µg/ml) in a TRIS buffer (20 mmol/L, pH 7.4, 37oC), containing EDTA (5 mmol/L), and

protease inhibitors (as above), and incubated (2h, 37oC) with [125I] ET-1 (20 pmol/L)

(Amersham Pharmacia Biotech, UK) in the absence and presence of ET-1 (0.002-20

nmol/L, American Peptide Co., USA), ET-3 (0.0001-200 nmol/L, American Peptide Co.,

USA) or A-192621 (0.00002-1 µmol/L, Abbott Laboratories, USA). Excess unlabelled

ET-1 (200 nmol/L) was used to measure non-specific binding (NSB) (9.4 ± 0.9%, n=22),

and total binding (TB) was determined in the absence of unlabelled ET peptide.

8

Receptor-bound 125I-ET-1 was separated from unbound after dilution with ice-cold TRIS

buffer (20 mmol/L) containing bovine serum albumin (2% w/v, Sigma, UK) and

bacitracin (0.1mmol/L). Separation occurred under vacuum filtration (Millipore, USA)

across glass microfibre filters (25 mm diameter) (Whatman, UK) and radioactivity on

each filter counted (Wallac 1410, Finland). Specific binding (SB) was calculated as TB-

NSB. Data were analysed by non-linear regression and fitted to a one or two site

model (Graphpad Prism), and regression analysis of the data was used to determine a

two site model when P<0.05.

Protein synthesis. Petri dishes (35mm diameter) were pre-incubated for 2 hours with

foetal calf serum (4% v/v) in M199. Aliquots of cell suspension (1 ml) were pipetted

gently onto Petri dishes, and after 1 h viable cardiomyocytes became attached to the

surface of the dish. The dishes were then washed with fresh CCT medium to remove

non-attached cells and cell debris and the attached cells were exposed for 24 h to l-U-

14C phenylalanine (0.1µCi/ml culture medium; Amersham Pharmacia Biotech, UK).

Incorporation of radioactivity into the acid-insoluble cell fraction was determined under

basal conditions and in the presence of ET receptor agonists/antagonists (8). The

attached cells were then washed with an aliquot (1 ml) of ice-cold PBS, prior to the

addition of an aliquot (1 ml) of ice-cold trichloroacetic acid (10% w/v). After storage

overnight at 4oC, the acid containing the intracellular precursor pool was removed from

the dishes and the attached cells were washed with an aliquot (1ml) of PBS. The

precipitate remaining on the culture dishes was dissolved in an aliquot (1 ml) of NaOH

(0.1 M)/sodium dodecyl sulphate (0.01% w/v) by overnight incubation at 37oC. In these

samples, concentration of DNA was determined by a spectrophotometric method in

which bisbenzamide dye was incorporated into DNA, and the radioactivity was counted.

9

The ratio of l-U-[14C] phenylalanine incorporated to DNA per culture served as a

measure of de novo synthesis of protein.

Contractile amplitude. Cardiomyocytes were subjected to field stimulation at 0.5 Hz with

biphasic pulses of 0.5 ms duration at 60 V under basal conditions and in the presence

of ET receptor agonists/antagonists. Cell shortening was assessed by video-edge

detection (Crescent Electronics, VED 104) (22), and data were digitised, recorded and

analysed using software provided by Dr. John Dempster (University of Strathclyde).

Data analysis. Data are expressed as means ± SE where n denotes number of rats in

which plasma ir-ET-1 was measured, or number of heart cell preparations used to

analyse gene expression, contractile amplitude, receptor binding or protein synthesis.

Statistical analyses were performed by analysis of variance to detect significant

differences for between group or within group effects and post-hoc comparisons by

Bonferroni or an unpaired Student’s t test as appropriate.

10

RESULTS

Expression. ET-1 concentration (pmol/L) was greater (P<0.05) in plasma of SHRs (3.98

± 1.3, n=4) than WKY rats (1.1 ± 0.1. n=7) at 8 weeks; thereafter, values were similar

between strains. Plasma concentration did not alter with age in WKY rats (1.1 ± 0.1,

n=7, 8 weeks v 0.9 ± 0.1, n=3, 24 weeks. PreproET-1 mRNA expression was greater in

SHR than WKY cardiomyocytes at 20 and 24 weeks (P<0.05); ECE mRNA was not

different between SHR and WKY cells at any age (Fig. 1, A and B). Expression of

cardiomyocyte ETA and ETB receptor mRNAs increased from 12 to 20 weeks in both

strains but were not different between strains (Fig. 1, C and D).

Homologous binding. A one site model was obtained following non-linear regression of

data from each experiment (Fig. 2, A and B). In cardiomyocyte membranes of SHRs at

12 weeks, ET receptor number was greater (P<0.05) than that of WKY rats, whereas at

20 weeks, receptor number had declined to that of WKY rats. Affinity (Kd) of ET-1 for

ET receptors did not decrease significantly between 12 and 20 weeks and was similar

between strains at each age.

Heterologous binding. A two site model was obtained following non-linear regression of

data from each experiment using ET-3 to displace 125I-ET-1 binding to cardiomyocyte

membranes from SHRs at 12, 16 and 20 weeks and from WKY rats at 20 weeks (Fig. 3,

A-C). IC50 values are given in Table 1. The proportion of high affinity binding sites was

22-25%. The identities of the high (pmol/L) and low affinity (nmol/L) binding sites for

ET-3 were confirmed as the ETB and ETA receptor, respectively, in heterologous

competition experiments using the ETB receptor-selective antagonist, A-192621 (data

not shown). In contrast, a one site model was obtained following non-linear regression

of data from WKY rats at 12 and 16 weeks, indicating that only the nmol/L affinity

11

binding site was present on these cells. The affinity of ET-3 for the ETB receptor (pmol/L

site) in SHR membranes increased (P<0.05) with disease progression and was greater

(P<0.05) than that of WKY rats at 20 weeks. The affinity of ET-3 for the ETA receptor

(nmol/L site) decreased (P<0.05) with age in WKY rats.

Hypertrophic function. ET-1 stimulated (P<0.05) protein synthesis in SHR and WKY rat

cardiomyocytes at 12, 16 and 20 weeks (Fig. 4). The response to ET-1 (10-9 mol/L)

was not altered with age or strain. S6c (10-7mol/L), increased (P<0.05) protein

synthesis in SHR but not WKY rat cardiomyocytes at each age (Fig. 4). A-192621, at a

concentration selective for interaction with ETB receptors (10-10 mol/L), did not alter

basal protein synthesis in WKY cardiomyocytes at any age, but paradoxically displayed

agonist activity (P<0.05) per se in SHR cardiomyocytes at 12 weeks and, markedly, at

16 and 20 weeks (Fig. 5). In the presence of A-192621 (10-10 mol/L), the response to

ET-1 (10-9 mol/L) was attenuated (P<0.05) in SHR cardiomyocytes at 12 weeks, but not

at 16 and 20 weeks nor in WKY cardiomyocytes at any age (Fig. 6). Similarly, the

response to S6c in SHR cardiomyocytes was abolished by A-192621 (10-10 mol/L) at 12

weeks but not at 16-20 weeks.

Contraction. EC50 values (range: 4.6x10-11 mol/L to 1.8x10-10 mol/L) and contraction

maxima (range 38.0-50.8% increase from basal) for ET-1 did not alter with age in SHR

cardiomyocytes (data not shown). The response to a sub-maximal concentration of ET-

1 (10-9 mol/L) was (i) not altered between strain, (ii) abolished by the ETA receptor

selective antagonist, ABT-627 (10-9 mol/L), but (iii) not altered by the ETB receptor

selective antagonist, A-192621 (10-7 mol/L) in WKY or SHR cardiomyocytes (8-24

weeks). S6c (10-7 mol/L) did not affect cell shortening in either strain at any age.

12

DISCUSSION

This study has provided evidence that ETB receptors are already present on

cardiomyocytes of SHRs prior to development of LVH but are absent from

cardiomyocytes of normotensive WKY rats. Furthermore, the pharmacological

characteristics of these receptors become altered during progression of cardiomyocyte

hypertrophy in vivo. Taken together, these findings have implications for the role of

endogenous endothelin signalling pathways, and the contribution of ETB receptors

specifically, during the development of LVH.

Increased plasma levels of ETs are positively correlated with severity of LVH in man

(15). There are conflicting data however regarding whether plasma ET-1 levels are

elevated in SHRs. Thibault et al. found elevated levels at 18 weeks when moderate

LVH was evident (39); others (23, 26), including ourselves, detected no change. In

contrast to Kohno et al. (23) who reported normal levels in pre-hypertensive SHRs at 6

weeks, we observed a transient increase in plasma ET-1 levels at 8 weeks, subsequent

to the onset of hypertension and preceding development of LVH, suggesting a possible

association. However, ET-1 has a short half-life (4-7 minutes) (30) which may limit the

influence of plasma-derived peptide directly upon the myocardium. The mixed ETA/B

antagonist, bosentan, causes some regression of LVH without reducing blood pressure,

indicating that locally-derived ET may exert a direct influence on cardiomyocyte

hypertrophy independently of the peptide’s pressor effects (20). Iyer et al. reported

increased ET-1 content in hearts of SHRs at 8 but not 4 weeks relative to WKY rats

(18); others detected no change at 10-12 weeks (5) and 18 weeks (39). Increased

mechanical stretch and paracrine mediators induce expression of preproET-1 mRNA in

neonatal cardiomyocytes in vitro (38, 43). Although hypertension in vivo might be

expected initially to increase mechanical stretch of cardiomyocytes, we found no

13

differences in preproET-1 or ECE mRNA expression between strains before onset of

LVH. Secretion of ET-1 from cardiac mesothelial cells is enhanced in SHRs at 9 weeks

(25); non-myocytes could provide an alternative source of ET-1 to initiate LVH (41).

ET receptor number was greater in cardiomyocyte membranes of SHRs than WKY rats

at 12 weeks. ET receptor number is also elevated early in development of LVH upon

aortic banding in rats (3), supporting early recruitment of ET-dependent signalling

mechanisms following pressure overload. In contrast, others have reported decreased

ET-1 binding site density in ventricular membranes from SHRs at 10-14 weeks (5, 14)

compared to WKY rats. At 20 weeks, when compensated LVH was present, receptor

number declined to values similar to or lower than those of WKY membranes, in

agreement with the decrease reported by Gu et al at 25 weeks (14). Crude ventricular

membrane preparations may not however accurately reflect changes occurring directly

in cardiomyocytes since non-myocytes possess a high density of ET receptors (21).

Cardiomyocyte ETA and ETB receptor mRNA expression was similar between SHRs

and WKY rats at all ages indicating that post-transcriptional mechanisms account for

differences between strains in relative abundance of receptor subpopulations. This

contrasts with the finding of Kanno et al. (19) that ETB receptor mRNA expression is up-

regulated in hypertrophying neonatal cardiomyocytes in vitro and raises questions

regarding extrapolation from neonates to adults. Inability to detect ETB receptors on

cardiomyocytes of WKY rats at 12 and 16 weeks agrees with the study of Fareh et al.

who reported that >90% of ET-1 binding sites on ventricular cardiomyocyte membranes

from young adult Sprague-Dawley rats were of the ETA subtype (13). Although Thibault

et al reported ratios of 25% ETB: 75%ETA in both strains at 18 weeks (39), crude

ventricular membranes were used, so cellular localisation of each receptor

14

subpopulation cannot be ascertained. While the total number of ET-1 binding sites

present on SHR cardiomyocytes was lower at 20 than at 12 weeks, the proportion of

each subtype did not change indicating that both subpopulations are down-regulated

with progression from onset to attainment of compensated LVH. Greater ET-1 binding

site density in WKY cardiomyocytes at 20 than 12 weeks could reflect the appearance

of ETB receptors in this strain with advancing age, occurring later than in SHRs. This is

consistent with the proposition that LVH following pressure overload represents

accelerated myocardial aging.

In contrast to the hypertrophic response elicited in WKY cells, which was almost

exclusively attributable to ETA receptor stimulation, the majority of the response initiated

in SHR cardiomyocytes at 12 weeks was associated with ETB receptor involvement,

since sarafotoxin 6c elicited a similar response to ET-1, even though ETB receptors

accounted for only ~25% of ET receptors present. Involvement of both receptor

subtypes in initiating hypertrophy of neonatal (16, 38) and adult cardiomyocytes (8) has

been demonstrated in vitro, even though the proportion of ETB receptors present on

cardiomyocytes obtained from healthy rats is negligible (<10%) (13).The number of

receptors present does not necessarily imply involvement in, or relate to efficacy of a

particular response; this highlights the importance of combining investigation of binding

characteristics with appropriate functional bioassays. Reduced receptor number in SHR

cells at 20 weeks was not associated with decreased maximum response to ET-1,

indicating the presence of surplus receptors.

The homologous competition data obtained at 12 weeks indicate that ET-1 binds with

high affinity (pmol/L) to cardiomyocyte ET receptors, representing binding

predominantly to ETA receptors in SHR, and exclusively so in WKY. It is likely that

15

levels of ET-1 in the vicinity of healthy cardiomyocytes would be significantly less than

the Kd value of 2x10-10 mol/L reported (13, 23, 26). However the presence of ETB

receptors on SHR cardiomyocytes would enable picomolar levels of ET-3, which would

not interact significantly with ETA receptors, to act in concert with ET-1 in initiating LVH;

indeed ET-3 is secreted by non-myocytes and initiates hypertrophy of neonatal

cardiomyocytes in vitro (38, 41, 43).

The affinity of ETB receptors present on SHR cells was enhanced with disease

progression. Such changes have implications for modulation of cell responsiveness to

the growth-effects of ET-1 and ET-3 during development of LVH in vivo since significant

stimulation might be achieved via ETB receptor-mediated signalling mechanisms to

initiate and maintain hypertrophic growth even in the absence of elevated peptide

levels. Enhanced affinity might be attributed to post-translational modification of the

receptor protein. The antagonist, A-192621, which binds selectively to ETB receptors

(8), paradoxically displayed agonist activity in SHR cells. This observation is also

compatible with specific structural changes to the receptor protein resulting in altered

intrinsic efficacy of the A-192621-receptor complex. A-192621 displayed partial agonist

activity at 12 weeks and partially attenuated the response to the full agonists, ET-1 and

S6c, but acquired almost full agonist activity at 16-20 weeks, such that A-192621 no

longer antagonised the responses to ET-1 and S6c. Changes to receptor

characteristics mainly occurred between 12 and 16 weeks, corresponding to the onset

and early progression of hypertrophic growth of the SHR myocardium (4); further

increases in the affinity of ET-3 for the ETB receptor and in the activity of A19261 were

marginal at 20 weeks. Structural alterations might be accounted for by oxidation of

amino, thiol, diazo and tyrosyl groups. Evidence is emerging to support a role for

oxidative stress and its interaction with ET-1, in development of LVH (17, 34). Oxidative

16

stress can influence binding of aldosterone to mineralocorticoid receptors (33). Lipid

peroxidation modifies plasmalemmal proteins (12), and enhances the opening

probability of SR ryanodine receptors (1), and calmodulin binding to calcium release

channels in skeletal muscle (46); attenuated dopamine D1A receptor-effector coupling

has been attributed to lipid peroxidation of receptors in the proximal renal tubule of

SHRs (42).

The positive contractile effect of ET-1 was exclusively attributed to ETA receptor

activation in both SHR and WKY cells. It is unclear whether levels of ET-1 present in

the vicinity of the cardiomyocytes (23, 26, 39) would enable the peptide to elicit an

inotropic response in vivo even in hypertrophying SHR myocardium. In contrast to that

of ETB receptors, the affinity of ETA receptors was not enhanced with disease

progression. The maximum response to ET-1 was constant with age in SHR cells and

was not different to that of WKY cells, confirming the finding of Delbridge et al. at 12

weeks (10), despite reduced Bmax values in SHR cells at 20 weeks compared to 12

weeks. These data support the presence of ‘spare’ ETA receptors.

In conclusion, the presence of ETB receptors before onset of cardiomyocyte

hypertrophy, coupled with responsiveness to sarafotoxin 6c of SHR but not WKY cells,

support the involvement of ETB receptors in initiating cardiomyocyte hypertrophy

following pressure overload, while altered ETB receptor characteristics during active

hypertrophic growth indicate that ETB receptor-dependent mechanisms may also

contribute to disease progression. These findings indicate a more prominent role for

ETB receptors than previously envisaged in the pathogenesis of LVH, and have

important implications for the current debate regarding the choice of receptor subtype

17

selective or non-selective endothelin antagonists for therapeutic intervention. Early

intervention with ETB receptor-selective antagonists may be beneficial in preventing or

retarding development of LVH in hypertensive patients although this benefit might be

offset by attenuated ETB-mediated vasodilatation and hence exacerbate already

elevated blood pressure; intervention studies with ETB receptor-selective antagonists in

SHRs and other experimental models are now clearly warranted.

Acknowledgements

This study was funded by the British Heart Foundation (PG: 1999150). The authors

express thanks to Dr J. Wessale (Abbott Laboratories) for the gift of the ET receptor

antagonists, ABT-627 and A-192621.

18

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FIGURE LEGENDS

Fig. 1: Expression of preproET-1 (a), ECE (b), ETA receptor (c) and ETB receptor (d)

mRNA in SHR and WKY rat ventricular cardiomyocytes at 8-24 weeks. Band intensity is

expressed as net intensity relative to GAPDH mRNA. Data are expressed as mean ±

SE of 7 preparations. *P<0.05 vs age-matched WKY rats.

Fig. 2: Homologous competition by unlabelled ET-1 of 125I-ET-1 binding to ET receptors

present on sarcolemmae prepared from ventricular cardiomyocytes of SHRs and WKY

rats at 12 (a) and 20 weeks (b). Specific binding (SB %), Kd and Bmax values represent

mean ± SE of 3 experiments. *P<0.05 vs age-matched WKY rats. † P<0.05 vs SHR at

12 weeks.

Fig. 3: Heterologous competition by unlabelled ET-3 of 125I-ET-1 binding to ET

receptors present on sarcolemmae prepared from ventricular cardiomyocytes of SHR

and WKY rats at 12 (a), 16 (b) and 20 weeks (c). Specific binding (SB %) represents

the mean ± SE of 3-4 experiments.

Fig. 4: Effects of ET-1 (10-9 mol/L) and S6c (10-7 mol/L) on incorporation of l-U[14C]

phenylalanine into protein in ventricular cardiomyocytes, isolated from SHRs and WKY

rats at 12, 16 and 20 weeks, and maintained in culture (24 h). Radioactivity

incorporated was corrected for DNA content, as an index of cell number. Data are

percentage differences from basal and represent mean ± SE of 8 preparations. *P<0.05

vs age-matched WKY rats. +P<0.05 vs basal response.

27

Fig. 5: Effects of A-192621 (10-10 mol/L) on incorporation of l-U[14C] phenylalanine into

protein in ventricular cardiomyocytes, isolated from SHRs and WKY rats at 12, 16 and

20 weeks, and maintained in culture (24 h). Radioactivity incorporated was corrected

for DNA content, as an index of cell number. Data are percentage differences from

basal and represent mean ± SE of 10 preparations. *P<0.05 vs response in the

absence of ligand.

Fig. 6: Effects of A-192621 (A192; 10-10 mol/L) on incorporation of l-U[14C]

phenylalanine into protein in the presence of ET-1 (10-9 mol/L) and S6c (10-7 mol/L),

respectively, in ventricular cardiomyocytes, isolated from SHRs and WKY rats at 12 (a-

b), 16 (c-d) and 20 (e-f) weeks, and maintained in culture (24 h). Radioactivity

incorporated was corrected for DNA content, as an index of cell number. Data are

percentage differences from basal and represent mean ± SE of 3-6 preparations.

*P<0.05 vs basal response.

Fig. 7: Effect of ET-1 (10-9 mol/L) (a), S6c (10-7 mol/L) (b), ET-1 (10-9 mol/L) in the

presence of ABT-627 (10-10 mol/L) and A-192621 (10-10mol/L) (c-d) on contractile

amplitude of ventricular cardiomyocytes isolated from SHRs and WKY rats at 8-24

weeks. Cell shortening is expressed as a percentage (δL%) of resting length (L). Data

are mean ± SE of 3-6 preparations.

28

8 12 16 20 24

WKYSHR

40

60

80

100

0

*

*

ET-1

Time (weeks)

Net

Inte

nsit

yre

lati

veto

GA

PD

H(%

)

8 12 16 20 24

WKYSHR

40

60

80

100

0

ECE

Time (weeks)

Net

Inte

nsi

tyre

lati

veto

GA

PD

H(%

)

8 12 16 20 24

0

25

50

75

WKYSHR

Time (weeks)

Net

Inte

nsit

yre

lati

veto

GA

PD

H(%

)

ETA

8 12 16 20 24

0

50

100

150

200

WKYSHR

Time (weeks)

Net

Inte

nsit

yre

lati

veto

GA

PD

H(%

)

ETB

(a) (b)

(c) (d)

Figure 1

29

-12 -11 -10 -9 -8

0

20

40

60

80

100

WKY

SHR

12 week WKY SHRKd (pmol/L) 205±42 237±28

Bmax (fM/mg protein ) 56±13 118±4*

Log10 [ET-1]

%[12

5I]

-ET

-1S

pec

ific

Bin

din

g

-12 -11 -10 -9 -8

0

20

40

60

80

100

WKY

SHR

Log10 [ET-1]

%[12

5I]

-ET

-1S

pec

ific

Bin

din

g

20 week WKY SHRKd (pmol/L) 335±19 311±43

Bmax (fM/mg protein) 83±6 75±7§

(a)

(b)

Figure 2

30

12 week

-12.5 -11.5 -10.5 -9.5 -8.5 -7.5 -6.5

0

20

40

60

80

100

WKYSHR

Log [ET-3]

%[1

25I]

-ET

-1S

pec

ific

Bin

din

g

16 week

-13 -12 -11 -10 -9 -8 -7

0

20

40

60

80

100

WKYSHR

Log [ET-3]

%[1

25I]

-ET

-1S

pe

cif

icB

ind

ing

20 week

-12.5 -11.5 -10.5 -9.5 -8.5 -7.5 -6.5

0

20

40

60

80

100

WKYSHR

Log [ET-3]

%[12

5I]

-ET

-1S

pe

cifi

cB

ind

ing

(a)

(b)

(c)

Figure 3

31

0

5

10

15

20

25

30

35 WKYSHR

12 16 20 12 16 20

------S6c ------

------ET-1 ------

**

10-9M

10-7M

++

+

+

+

+

+

+

+

+

Age (weeks)

l-U

-[14

C]-

ph

enyl

alan

ine

inco

rpo

rati

on

(%o

ver

bas

alva

lue)

Figure 4

32

-5

0

5

10

15

20 WKYSHR

*

* *

-----12----- -----16----- -----20-----

Age (weeks)

l-U

-[14

C]p

heny

lala

nine

inco

rpor

atio

n(%

over

basa

lval

ue)

Figure 5

33

A-192 ET-1 A-192+ET-1-10

0

10

20

30

40

*

*

12 week

* *

P=0.007

P=0.001 P=0.01

l-U

-[14

C]-

ph

enyl

alan

ine

inco

rpo

rati

on

(%o

ver

bas

alva

lue)

*

A-192 Sfx A-192+Sfx-10

0

10

20

30

40

WKY SHR

P=0.002 P=0.01

*

*

*

12 week

l-U-[

14C

]-p

hen

ylal

anin

ein

corp

ora

tio

n(%

ove

rb

asal

valu

e)

A-192 ET-1 A-192+ET-1-10

0

10

20

30

40

WKY SHR

*

*

16 week

*

P<0.025

l-U

-[14

C]-

ph

enyl

alan

ine

inco

rpo

rati

on

(%o

ver

bas

alva

lue)

A-192 Sfx A-192+Sfx-10

0

10

20

30

40 WKYSHR

*

*

*

16 week

l-U

-[14

C]-

ph

enyl

alan

ine

inco

rpo

rati

on

(%o

ver

bas

alva

lue)

A-192 ET-1 A-192+ET-1

-10

0

10

20

30

40

WKY SHR

*

*

20 week

* *

P<0.0001

l-U

-[14

C]-

ph

enyl

alan

ine

inco

rpo

rati

on

(%o

ver

bas

alva

lue)

A-192 Sfx A-192+Sfx

-10

0

10

20

30

40

* *

20 week

l-U

-[14

C]-

ph

enyl

alan

ine

inco

rpo

rati

on

(%o

ver

bas

alva

lue)

(b)(a)

(c) (d)

(e) (f)

Figure 6

34

12 16 20 24

0

50

100

150

200

WKYSHR

Age (weeks)

�

L(%

chan

gefr

omba

sal)

Sfx

8 12 16 20 24

0

50

100

150

200

ET-1 + ABT-627ET-1

ET-1 + A-192621

WKY

Age (weeks)

�

L(%

chan

gefr

omba

sal)

8 12 16 20 24

0

50

100

150

200 ET-1ET-1 + ABT-627

ET-1 + A-192621

SHR

Age (weeks)

�

L(%

chan

gefr

omba

sal)

8 12 16 20 24

0

50

100

150

200

WKY

SHR

ET-1

Age (weeks)

�

L(%

chan

gefr

om

bas

al)

(a) (b)

(c) (d)

Figure 7

35

Table 1

IC50 values determined from non-linear regression analysis of heterologous competitive binding (n=3-5).

WKY SHR

pM (ETB) site nM (ETA) site pM (ETB) site nM (ETA) site un-treated 8 week nd nd 250±12 4.45±0.21 12 week none 1.90±0.1 140±7 3.82±0.97 16 week none 2.72±0.05 11.5±7* 2.53±0.23 20 week 123±19 5.32±0.53* 8.3±3** 3.95±0.85 * P<0.05 between preceding age within strain; ** P<0.05 between strain (equivalent age-matched site); nd not determined