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Am J Physiol Heart Circ Physiol 287: H425-H432, 2004. First published February 26, 2004; doi:10.1152/ajpheart.00461.2003
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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, and Barbara J. McDermott

Department of Therapeutics and Pharmacology, Centre for Cardiovascular and Genetics Research, School of Medicine, Queen's University of Belfast, Belfast BT9 7BL, Northern Ireland, United Kingdom

Submitted 22 May 2003 ; accepted in final form 20 February 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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 before onset (8 and 12 wk) and during progression (16, 20, and 24 wk) of LVH and compared with 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 wk. ET binding-site density was twofold greater in SHR than WKY cells at 12 wk (P < 0.05) but normalized at 20 wk. ETB receptors were detected on SHR cardiomyocytes as early as 8 wk and their affinity increased progressively with age (P < 0.05), whereas ETB receptors were not detected on WKY cells until 20 wk. ET-1 stimulated protein synthesis with similar maximum responses between strains (21–30%), in contrast with sarafotoxin 6c, which stimulated protein synthesis in SHR (13–20%) but not WKY cells at 12–20 wk. 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 wk) coupled with functional responsiveness of SHR cells but not WKY cells to sarafotoxin 6c at 12 wk supports the involvement of ETB receptors before the onset of cardiomyocyte hypertrophy, whereas altered ETB receptor characteristics during active hypertrophy (16–24 wk) indicate that ETB receptor mechanisms may also contribute to disease progression.

spontaneously hypertensive rats; pressure overload; endothelin receptor

CONCENTRIC LEFT VENTRICULAR hypertrophy (LVH) occurs after pressure overload and is associated with thickening of the ventricular wall to normalize wall stress but ultimately leads to mechanical dysfunction and failure (24). Increased cardiac mass is attributed to increased mass of individual cardiomyocytes, proliferation of nonmyocytes, and synthesis of extracellular matrix (36, 40). Epidemiological studies indicate that regression of LVH with antihypertensive agents improves prognosis (27). Such treatments, however, only partially regress LVH; involvement of nonhemodynamic factors has also been postulated (9).

Endothelin (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 occur during hypertension and heart failure and correlate with severity of LVH (15). ET receptor antagonists attenuate LVH in some experimental models in vivo (17, 20). It is unclear whether this occurs as a direct result of ET receptor blockade on cardiomyocytes or represents an indirect effect that is 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 remodeling (3, 6, 35, 45). The actions of ET are mediated by ETA and ETB receptors, which are both present in heart (13, 39): ETA receptors have greater affinity for ET-1 than ET-3, whereas ETB receptors bind these peptides with equal affinity (2). Sarafotoxin 6c (S6c) is an ETB receptor-selective agonist; BQ-123 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 of ETA receptors, whereas 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 of ETB receptors (8, 17). ET-1 elicits a positive inotropic effect on the 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 upregulated in hypertrophying neonatal cardiomyocytes (19). ET-3 and mechanical stretch induce expression of preproET-1 mRNA, whereas hypertrophy of neonatal cardiomyocytes by each stimulus in vitro is attenuated by BQ-123 (38, 43). Nonmyocytes 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 because the initial attenuation by BQ-123 of the onset of LVH after aortic banding of adult rats is not sustained (17). These data highlight the importance of longitudinal studies that utilize 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 and 20 wk. Despite severe elevations of systemic arterial pressure, cardiac output is maintained initially by moderate LVH. Because alterations in cardiac performance may reflect many influences (intrinsic muscle properties, loading conditions, altered systemic and/or coronary hemodynamics), studies in cardiomyocytes specifically are useful to dissect out adaptations intrinsic to them from those of fibrosis and nonmyocyte proliferation. We have characterized SHRs comprehensively at the cardiomyocyte level allowing precise application of this model in investigations of pathogenetic mechanisms; hypertension is followed by active hypertrophic growth between 16 and 20 wk. This is evidenced by increased cell mass and width, which subsequently decelerate at 24 wk 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 overexpression 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 on isolated cardiomyocytes (10).

The aim of this longitudinal study was to investigate whether alterations in the ET receptors are initiated in cardiomyocytes before the onset of LVH in SHRs, and if so, whether these alterations are associated with the development and progression of ventricular cell hypertrophy. Appropriate comparisons were made using cardiomyocytes isolated from age-matched normotensive Wistar-Kyoto (WKY) rats.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental model. Male WKY rats and SHRs were obtained from Harlan at 4 wk and maintained until sampling at 8, 12, 16, 20, and 24 wk 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. After administration of deep anesthesia to rats using isoflurane (Abbott Laboratories), the hearts were rapidly excised and placed in ice-cold saline, and blood was collected from the chest cavity into ice-cold tubes containing EDTA (2 mmol/l) and aprotinin (500 IU/l; Sigma Chemical). Immunoreactive ET was extracted from plasma using C18 Sep-Pak cartridges (Waters Associates) and measured by RIA (Phoenix Pharmaceuticals; Ref. 18).

Cardiomyocyte isolation. Excised hearts were cannulated through the ascending aorta, and ventricular cardiomyocytes were isolated by enzymatic digestion (collagenase, 0.4 mg/ml) using Langendorff perfusion (8). After purification, cells were suspended at 1.5 x 105 viable cardiomyocytes/ml in a creatinine-carnitine-taurine (CCT) medium that consisted of modified glutamine-free medium 199 (M199) supplemented with Earle's salts (GIBCO), 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 {beta}-D arabinofuranoside (10 µM) to prevent growth of nonmyocytes (Sigma; Ref. 8).

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 synthesized from 2 µg of total RNA by reverse transcriptase (Reverse-iT kit; Abgene). Gene-specific primers were based upon those previously reported (37). After initial denaturation at 94°C for 4 min, cycling profiles included specific annealing temperatures and cycle numbers [preproET-1: 54°C, 31; endothelin-converting enzyme (ECE): 60°C, 28; ETA/ETB receptors: 55°C, 32; respectively], followed by extension at 72°C for 60 s. PCR products were electrophoresed on 2% agarose gels and stained with ethidium bromide. Gels were visualized under ultraviolet illumination and analyzed using a Gene Genius Gel documentation system with Gene Tools analysis software (Syngene). Band intensity was expressed as the ratio of target mRNA to GAPDH mRNA.

Preparation of sarcolemmae. Viable cardiomyocytes were suspended in a HEPES (20 mmol/l) buffer that contained the protease inhibitors aprotinin (0.8 µmol/l), bacitracin (0.1 mmol/l), benzamidizine (0.1 mmol/l), EDTA (5 mmol/l), leupeptin (2 µmol/l), and PMSF (0.1 mmol/l) (all from Sigma), and were homogenized at 9,500 rpm (Ultra-Turax-T25; Janke and Kunkel) for 30 s. Disrupted cells were centrifuged (2,000 rpm for 5 min at 4°C; Mistral MSE 400) to sediment cell nuclei and mitochondrial fractions, and supernatants were then centrifuged three times at 20,000 rpm for 30 min at 4°C. The pellets were stored at –70°C.

Homologous/heterologous competition binding. Sarcolemmae were suspended (20 µg/ml) in a Tris buffer (20 mmol/l, pH 7.4, 37°C) that contained EDTA (5 mmol/l) and protease inhibitors (as above) and incubated (for 2 h at 37°C) with [125I]ET-1 (20 pmol/l; Amersham Pharmacia Biotech) in the absence and presence of ET-1 (0.002–20 nmol/l; American Peptide), ET-3 (0.0001–200 nmol/l; American Peptide), or A-192621 (0.00002–1 µmol/l; Abbott Laboratories). Excess unlabeled ET-1 (200 nmol/l) was used to measure nonspecific binding (NSB; 9.4 ± 0.9%; n = 22), and total binding (TB) was determined in the absence of unlabeled ET peptide. Receptor-bound [125I]ET-1 was separated from unbound after dilution with ice-cold Tris buffer (20 mmol/l) that contained bovine serum albumin (2% wt/vol; Sigma) and bacitracin (0.1 mmol/l). Separation occurred under vacuum filtration (Millipore) across glass microfiber filters (25 mm diameter; Whatman) and radioactivity on each filter was counted (Wallac 1410). Specific binding (SB) was calculated as TB – NSB. Data were analyzed by nonlinear 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 (35 mm diameter) were preincubated for 2 h with fetal calf serum (4% vol/vol) 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 nonattached cells and cell debris, and the attached cells were exposed for 24 h to L-U-[14C]phenylalanine (0.1 µCi/ml of culture medium; Amersham Pharmacia Biotech). 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 a 1-ml aliquot of ice-cold PBS before the addition of a 1-ml aliquot of ice-cold trichloroacetic acid (10% wt/vol). After storage overnight at 4°C, the acid containing the intracellular precursor pool was removed from the dishes, and the attached cells were washed with a 1-ml aliquot of PBS. The precipitate remaining on the culture dishes was dissolved in a 1-ml aliquot of 0.1 M NaOH-SDS (0.01% wt/vol) by overnight incubation at 37°C. In these samples, concentration of DNA was determined by a spectrophotometric method in which bis-benzamide dye was incorporated into DNA and the radioactivity was counted. The ratio of L-U-[14C]phenylalanine incorporated into DNA per culture served as a measure of de novo protein synthesis.

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 (VED 104; Crescent Electronics; Ref. 22), and data were digitized, recorded, and analyzed using WCP for Windows software (version 1.8) provided by Dr. John Dempster (University of Strathclyde).

Data analysis. Data are expressed as means ± SE, where n denotes the number of rats in which plasma immunoreactive ET-1 was measured or the number of heart cell preparations used to analyze gene expression, contractile amplitude, receptor binding, or protein synthesis. Statistical analyses were performed by ANOVA 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.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Expression. ET-1 concentration (in 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 wk; thereafter, values were similar between strains. Plasma concentration did not alter with age in WKY rats (1.1 ± 0.1; n = 7 at 8 wk vs. 0.9 ± 0.1; n = 3 at 24 wk). PreproET-1 mRNA expression was greater in SHR than WKY cardiomyocytes at 20 and 24 wk (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 wk in both strains but were not different between strains (Fig. 1, C and D).



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  		Fig. 1. Expression of preproendothelin-1 (preproET-1; A), ECE (B), endothelin (ET)A receptor (C), and ETB receptor (D) mRNA in ventricular cardiomyocytes from spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) rats at 8–24 wk. Band intensity is expressed as net intensity relative to GAPDH mRNA. Data are expressed as means ± SE of 7 preparations; *P < 0.05 vs. age-matched WKY rats.

 
Homologous binding. A one-site model was obtained using nonlinear regression of data from each experiment (Fig. 2). In cardiomyocyte membranes of SHRs at 12 wk, ET receptor number was greater (P < 0.05) than that of WKY rats, whereas at 20 wk, 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 wk and was similar between strains at each age.



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  		Fig. 2. Homologous competition by unlabeled 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 (B) wk. Specific binding (Kd) and maximum binding (Bmax) values represent means ± SE of 3 experiments; *P < 0.05 vs. age-matched WKY rats; §P < 0.05 vs. SHRs at 12 wk.

 
Heterologous binding. A two-site model was obtained via nonlinear 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 wk and from WKY rats at 20 wk (Fig. 3). IC50 values are given in Table 1. The proportion of high-affinity binding sites was 22–25%. The identities of the high- (in pmol/l) and low- (in nmol/l) affinity binding sites for ET-3 were confirmed as the ETB and ETA receptors, respectively, in heterologous competition experiments using the ETB receptor-selective antagonist A-192621 (data not shown). In contrast, a one-site model was obtained using nonlinear regression of data from WKY rats at 12 and 16 wk, which indicated that only the low-affinity binding site was present on these cells. The affinity of ET-3 for the ETB receptor (the 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 wk. The affinity of ET-3 for the ETA receptor (the nmol/l site) decreased (P < 0.05) with age in WKY rats.



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  		Fig. 3. Heterologous competition by unlabeled 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 (C) wk. Specific binding represents means ± SE of 3 or 4 experiments.

 

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  		Table 1. IC50 values determined from nonlinear regression analysis of heterologous competitive binding

 
Hypertrophic function. ET-1 stimulated (P < 0.05) protein synthesis in SHR and WKY cardiomyocytes at 12, 16, and 20 wk (Fig. 4). The response to ET-1 (10–9 mol/l) was not altered with age or strain. S6c (10–7 mol/l) increased (P < 0.05) protein synthesis in SHR but not WKY 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 wk and, markedly, at 16 and 20 wk (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 wk but not at 16 and 20 wk 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 wk but not at 16–20 wk.



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  		Fig. 4. Effects of ET-1 (10–9 mol/l) and sarafotoxin 6c (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 wk 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 level and represent means ± SE of 8 preparations. *P < 0.05 vs. age-matched WKY rats; +P < 0.05 vs. basal response.

 


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  		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 wk 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 numbers and represent means ± SE of 10 preparations. *P < 0.05 vs. response in absence of ligand.

 


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  		Fig. 6. Effects of A-192621 (A-192; 10–10 mol/l) on incorporation of L-U-[14C]phenylalanine into protein in the presence of ET-1 (10–9 mol/l) and sarafotoxin 6c (Sfx; 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) wk 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 values and represent means ± SE of 3–6 preparations. *P < 0.05 vs. basal response.

 
Contraction. EC50 values (range, 4.6 x 10–11 to 1.8 x 10–10 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 submaximal concentration of ET-1 (Fig. 7; 10–9 mol/l) was 1) not altered between strains, 2) abolished by the ETA receptor-selective antagonist ABT-627 (10–9 mol/l), but 3) not altered by the ETB receptor-selective antagonist A-192621 (10–7 mol/l) in WKY or SHR cardiomyocytes (8–24 wk). S6c (10–7 mol/l) did not affect cell shortening in either strain at any age.



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  		Fig. 7. Effects of ET-1 (10–9 mol/l; A), sarafotoxin 6c (Sfx; 10–7 mol/l; B), and ET-1 (10–9 mol/l) in the presence of ABT-627 (10–10 mol/l) and A-192621 (10–10 mol/l; C, D) on contractile amplitude of ventricular cardiomyocytes isolated from SHRs and WKY rats at 8–24 wk. Cell shortening is expressed as a percentage ({delta}L%) of resting length (L). Data are means ± SE of 3–6 preparations.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study has provided evidence that ETB receptors are already present on cardiomyocytes of SHRs before 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 signaling 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 humans (15). There are conflicting data, however, regarding whether plasma ET-1 levels are elevated in SHRs. Thibault et al. (39) found elevated levels at 18 wk when moderate LVH was evident; others (23, 26), including ourselves, detected no change. In contrast with the work of Kohno et al. (23), who reported normal levels in prehypertensive SHRs at 6 wk, we observed a transient increase in plasma ET-1 levels at 8 wk subsequent to the onset of hypertension and preceding development of LVH, which suggests a possible association. However, ET-1 has a short half-life (4–7 min; Ref. 30), which may limit the influence of plasma-derived peptide directly on the myocardium. The mixed ETA/B antagonist bosentan causes some regression of LVH without reducing blood pressure, which indicates that locally derived ET may exert a direct influence on cardiomyocyte hypertrophy independently of the pressor effects of the peptide (20). Iyer et al. (18) reported increased ET-1 content in hearts of SHRs at 8 but not 4 wk relative to WKY rats; others detected no change at 10–12 wk (5) and 18 wk (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 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 wk (25); nonmyocytes 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 wk. ET receptor number was also elevated early in development of LVH on aortic banding in rats (3), which supports early recruitment of ET-dependent signaling mechanisms after pressure overload. In contrast, others have reported decreased ET-1 binding-site density in ventricular membranes from SHRs at 10–14 wk (5, 14) compared with WKY rats. At 20 wk, 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. (14) at 25 wk. Crude ventricular membrane preparations may not, however, accurately reflect changes occurring directly in cardiomyocytes because nonmyocytes 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, which indicates that posttranscriptional 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 upregulated 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 wk agrees with the study of Fareh et al. (13), who reported that >90% of ET-1-binding sites on ventricular cardiomyocyte membranes from young adult Sprague-Dawley rats were of the ETA subtype. Although Thibault et al. (39) reported ratios of 25% ETB to 75% ETA in both strains at 18 wk, crude ventricular membranes were used, so cellular localization of each receptor subpopulation cannot be ascertained. Although the total number of ET-1 binding sites present on SHR cardiomyocytes was lower at 20 than at 12 wk, the proportion of each subtype did not change, which indicates that both subpopulations are downregulated with progression from onset to attainment of compensated LVH. Greater ET-1 binding-site density in WKY cardiomyocytes at 20 than at 12 wk could reflect the appearance of ETB receptors in this strain with advancing age, which occurs later than in SHRs. This is consistent with the proposition that LVH after pressure overload represents accelerated myocardial aging.

In contrast with 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 wk was associated with ETB receptor involvement, because S6c elicited a similar response to ET-1 although ETB receptors accounted for only ~25% of ET receptors present. Involvement of both receptor subtypes in initiating hypertrophy of neonatal (17, 38) and adult (8) cardiomyocytes has been demonstrated in vitro, although the proportion of ETB receptors present on cardiomyocytes obtained from healthy rats is negligible (<10%; Ref. 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 wk was not associated with decreased maximum response to ET-1, which indicates the presence of surplus receptors.

The homologous competition data obtained at 12 wk indicate that ET-1 binds with high affinity (pmol/l) to cardiomyocyte ET receptors representing binding predominantly ETA receptors in SHR and predominantly so in WKY. It is likely that levels of ET-1 in the vicinity of healthy cardiomyocytes would be significantly less than the Kd value of 2 x 10–10 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 nonmyocytes 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, because significant stimulation might be achieved via ETB receptor-mediated signaling mechanisms to initiate and maintain hypertrophic growth even in the absence of elevated peptide levels. Enhanced affinity might be attributed to posttranslational 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 wk and partially attenuated the response to the full agonists ET-1 and S6c but acquired almost full agonist activity at 16–20 wk such that A-192621 no longer antagonized the responses to ET-1 and S6c. Changes to receptor characteristics mainly occurred between 12 and 16 wk, which corresponds to the onset and early progression of hypertrophic growth of the SHR myocardium (4); additional increases in the affinity of ET-3 for the ETB receptor and in the activity of A-19261 were marginal at 20 wk. 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 (16, 34). Oxidative stress can influence binding of aldosterone to mineralocorticoid receptors (33). Lipid peroxidation modifies plasmalemmal proteins (12) and enhances the opening probability of sarcoplasmic reticulum ryanodine receptors (1), and the binding of calmodulin 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, which confirms the finding of Delbridge et al. (10) at 12 wk, despite reduced values for maximal binding capacity in SHR cells at 20 wk compared with 12 wk. 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 S6c of SHR but not WKY cells support the involvement of ETB receptors in initiating cardiomyocyte hypertrophy after pressure overload, whereas 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 selective or nonselective endothelin antagonists in 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.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was funded by British Heart Foundation Project Grant 1999150 and PhD Studentship FS98012.


   ACKNOWLEDGMENTS
 
The authors express thanks to Dr. J. Wessale (Abbott Laboratories) for the gift of the ET receptor antagonists ABT-627 and A-192621.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. Bell, Dept. of Therapeutics and Pharmacology, Queen's Univ. of Belfast, Whitla Medical Bldg., 97 Lisburn Rd., Belfast BT9 7BL, Northern Ireland, UK (E-mail: d.bell{at}qub.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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