CLINICAL PERSPECTIVE

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 114/8/830    most recent CIRCULATIONAHA.105.577288v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhao, X.-S. Articles by Shohet, R. V. Search for Related Content PubMed PubMed Citation Articles by Zhao, X.-S. Articles by Shohet, R. V. Related Collections Apoptosis Cell signalling/signal transduction Genetically altered mice

(Circulation. 2006;114:830-837.)
© 2006 American Heart Association, Inc.

Molecular Cardiology

Endogenous Endothelin-1 Is Required for Cardiomyocyte Survival In Vivo

Xiao-Song Zhao, MD, PhD; Wentong Pan, MD; Raffi Bekeredjian, MD; Ralph V. Shohet, MD

From Internal Medicine, University of Texas Southwestern Medical Center, Dallas (X.-S.Z., W.P.); Department of Cardiology, University of Heidelberg, Heidelberg, Germany (R.B.); and Department of Medicine, John A. Burns School of Medicine, University of Hawaii, Honolulu (R.V.S.). This study was conducted at the University of Texas Southwestern Medical Center before Drs Bekeredjian and Shohet moved to their present addresses.

Correspondence to Ralph V. Shohet, Department of Medicine, BSB 211D, 651 Ilalo St, Honolulu, HI 96813. E-mail shohet{at}hawaii.edu

Received September 20, 2005; revision received June 12, 2006; accepted June 22, 2006.

	Abstract

Top Abstract Introduction Methods Results Discussion Conclusions References

 
Background— Endothelin-1 (ET-1) has potent vasoconstrictor and hypertrophic actions. Pharmacological antagonists of endothelin receptors attenuate cardiac hypertrophy, have been approved for treatment of pulmonary hypertension, and are under investigation for treatment of heart failure. To investigate the role of ET-1 in the heart, we created mice with cardiomyocyte deletion of ET-1.

Methods and Results— Mice with cardiomyocyte-specific deletion of ET-1 are phenotypically normal when young. Remarkably, as the mice age or when young animals are subjected to aortic banding, they develop an unexpected phenotype of progressive systolic dysfunction and cardiac dilation. Echocardiography, necropsy, histology, and molecular phenotype confirm a dilated cardiomyopathy. Terminal deoxynucleotidyl transferase–mediated dUTP nick-end-labeling analysis reveals greater abundance of apoptotic nuclei in the ET-1–deficient hearts. Transcriptional and Western analyses suggest enhanced tumor necrosis factor (TNF)–mediated apoptosis with increases in caspase-8 activity. These ET-1–deficient hearts also have diminished nuclear factor (NF)-B activity, resulting in diminution of downstream inhibitors of TNF signaling.

Conclusions— Local ET-1 gene expression is necessary to maintain normal cardiac function and cardiomyocyte survival in mice with both age and hemodynamic stress. This cardiac-protective effect is mediated by paracrine ET-1 modulation of TNF-related apoptosis, in part through upregulation of NF-B signaling.

Key Words: apoptosis • cardiomyopathy • endothelin • tumor necrosis factor • nuclear factor-kappa B

	Introduction

Top Abstract Introduction Methods Results Discussion Conclusions References

 
Endothelin-1 (ET-1) was first identified as a potent vasoconstrictor peptide secreted by endothelium.¹ It also is expressed in the heart and has subsequently been shown to have a direct influence on cardiac myocytes, including hypertrophic and inotropic effects.^2–5 Both ET-1 receptors are found on cardiac myocytes. The ET_A receptor is more abundant (90%) and has been considered more important for the cardiac effects of ET-1, but the ET_B receptor may be more responsive to physiological stress.⁶ Remarkably, ET-1 has recently been shown to improve the survival of cardiomyocytes⁷ through a reduction in susceptibility to apoptosis.

Clinical Perspective p 837

Genetic approaches have been applied to investigate the function of the endothelin axis. ET-1 and ET_A gene knockout (KO) mice die after birth from asphyxia because of malformation of the hypopharynx.^8,9 Endothelin-converting enzyme-1–deficient animals have a similar phenotype.¹⁰ Mice lacking ET-1, ET_A, or endothelin-converting enzyme-1 also display defects of arterial formation. These genetic models help establish the function of the endothelin axis in embryonic development, but they do not produce detectable phenotypes in cardiac muscle or clues to the function of ET-1 in the adult heart. To study the isolated effects of cardiac ET-1, we have used a mouse strain with cardiomyocyte-specific deletion of the portion of the ET-1 gene encoding the mature ET-1 peptide generated with Cre-lox technology.¹¹ This allows us to explore the isolated effects of cardiac ET-1 in an animal that has enjoyed the normal developmental functions of ET-1.

Many studies have suggested a role for the endothelin axis in congestive heart failure. In animal models of heart failure, treatment with pharmacological inhibitors of the endothelin receptors has been shown to ameliorate ventricular remodeling and to prolong life after infarction.¹² This has prompted efforts to examine the effect of these agents in patients with heart failure. However, contrary to expectations, trials of inhibitors of both or specific ET receptors have not produced the anticipated clinical benefits.¹³ Our model allows us to examine the local paracrine effect of ET-1 in the heart and may help to explain the absence of a clinical benefit with inhibitors of the ET-1 axis. If ET-1 plays an important role in preserving cardiac myocytes exposed to stress, either that encountered chronically through life or with increased afterload, then strategies that block the action of ET-1 in the heart may be counterproductive, particularly in patients with other stimuli for cardiac apoptosis.

	Methods

Top Abstract Introduction Methods Results Discussion Conclusions References

 
Reagents and Animals
All chemicals are from Sigma Chemical Co (St Louis, Mo) unless specified. Antibodies are from Cell Signaling (Danvers, Mass). Cardiac-specific ET-1 KO mice were generated with the loxP-Cre system.¹⁴ Briefly, mice with loxP-bracketed ET-1 allele were mated with cardiac -myosin heavy chain (MHC)–Cre transgenic mice. We refer to the ET-1^lox/lox genotype as "wild type" (WT) for simplicity in this report. The only difference between this strain and true WT mice is the presence of the loxP sites bracketing exon 2 of the ET-1 gene. Excision of this exon removes the region of the gene that encodes the mature ET-1 peptide in cardiac myocytes. Aortic banding and unsedated echocardiography were performed as described.^15,16 All procedures were approved by the University of Texas Southwestern Animal Care Committee.

Real-Time Polymerase Chain Reaction Assays
Real-time polymerase chain reaction (PCR) was run as described.¹⁷ Cyclophilin A was used as an internal control.

Terminal Deoxynucleotidyl Transferase–Mediated dUTP Nick-End-Labeling Assays
Paraffin-embedded sections and 4% formaldehyde–fixed cardiomyocytes were evaluated for apoptosis by means of terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) (DeadEnd Fluorometric TUNEL System, Promega, Madison, Wis) according to the manufacturer’s protocol. Counterstaining with propidium iodide indicated the location of nuclei. For tissue sections, apoptotic nuclei were counted on whole sections of both ventricles under high-power (x40) magnification. Apoptotic cardiomyocytes were counted in 3 random high-power (x40) fields. Propidium iodine–stained nuclei were counted from 6 random x40 fields. The total number of nuclei in the section was then calculated by adjusting the average number of nuclei per field for the surface area of the section (obtained with Scion Image 1.63 software).

Electrophoretic Mobility Shift Assay
Electrophoretic mobility shift assays (EMSAs) were performed with 100 µg nuclear protein following published methods.¹⁸ Nuclear factor-B (NF-B) consensus sequence (5'-AGT TGA GGG GAC TTT CCC AGG C-3'; Promega) was end labeled with [-³²P]ATP using polynucleotide kinase. In previous studies,^19,20 this sequence produced highly specific NF-B bands in EMSAs.

Cell Culture and Transient Gene Transfection
Primary neonatal rat cardiomyocytes were prepared as described.²¹ After 48 hours of incubation in serum-free medium, Lipofectamine 2000 (Invitrogen, Carlsbad, Calif) was used for transfection as suggested by the manufacturer. When the transfected cells were treated with ET-1 or bosentan, the agents were added 24 hours after transfection. The culture medium was changed every 12 hours, with the inclusion of ET-1, Bosentan, or TNF, until harvest.

Endogenous NF-B Activity Assays
A pNF-B–SEAP reporter system (BD Biosciences) was used to monitor NF-B activity in transfected cardiomyocytes in which a secreted form of the human placental alkaline phosphatase (SEAP) cDNA is driven by 4 tandem copies of NF-B consensus sequence (B₄) fused to a basic TATA-like promoter. After endogenous NF-B proteins bind to the B₄, transcription is induced and the reporter gene is activated. The expressed protein was secreted and analyzed with the BD Great EscAPe SEAP Chemiluminescence Detection Kit. CMV-Renilla luciferase reporter was used as internal control.

Statistical Analysis
Pairwise comparisons between groups were performed with the Student t test with homoskedasticity or heteroskedasticity according to the variance comparison results. Comparisons between multiple groups were evaluated with ANOVA. Values are reported as mean±SEM, and values of P<0.05 were considered statistically significant.

The authors had full access to the data and take full responsibility for their integrity. All authors have read and agree to the manuscript as written.

	Results

Top Abstract Introduction Methods Results Discussion Conclusions References

 
Physiological and Histological Evaluation of Cardiac-Specific Deletion of ET-1
Previous evaluation of the MHC-Cre⁺, loxP^+/+ mice by Southern blot analysis showed cardiac-specific deletion of ET-1 with detectable excision of a reporter transgene in >80% of cardiac myocytes; transcript analyses indicated a substantial decrease in ET-1 mRNA in isolated cardiomyocytes compared with WT littermates with an intact ET-1 gene.¹⁴ Young ET-1 KO animals had normal mendelian distribution in litters, as well as normal growth, activity, and fertility. Cardiac function assessed by echocardiography was similar between the 2 groups of animals. Histology showed normal structure and organization of cardiac muscle fibers. Cardiac mass, indexed to body weight, was similar, and the histological surface area of cardiomyocytes was identical between ET-1 KO and WT animals.

Aging ET-1 KO Mice Exhibited Impaired Cardiac Function
At 2 months of age, the ET-1 KO and WT animals had indistinguishable left ventricular (LV) function by fractional shortening or area fractional shortening (Table I, available online in the accompanying Data Supplement). However, at 6 months, the ET-1–deleted mice had a significantly lower fractional shortening (although still within normal limits) compared with their WT sibs. Over the ensuing months, the left ventricles of ET-1 KO mice dilated and LV systolic function declined (Figure 1). Ultimately, the ET-1 KO mice died at much younger ages. The median life expectancy of ET-1 KO mice was 11 months (Figure 2), whereas WT mice live a normal lifespan of &2 years.

View larger version (37K): [in this window] [in a new window]   Figure 1. Echocardiographic determination of LV function. End-systolic diameter, end-diastolic diameter, and LV fractional shortening were measured on M-mode images from conscious animals (A–C). D, Representative M-mode images from WT and KO animals. P<0.05 for all comparisons between WT and KO after 7 months; n=6 to 10 in each group.

View larger version (14K): [in this window] [in a new window]   Figure 2. Survival curve of WT and KO mice.

Histological Evaluation
Histological characterization of the ET-1 KO hearts revealed dilated heart chambers with heterogeneity of myocyte size, including hypertrophy. Trichrome stains showed increased fibrosis (Figure 3).

View larger version (39K): [in this window] [in a new window]   Figure 3. Histological evaluation of dilated cardiomyopathy. Top, Four-chamber views of hearts with premortem fractional shortening noted below. Top microscopic images (hematoxylin and eosin [H&E] staining, x40) shows heterogeneity and hypertrophy of cardiomyocytes in a KO heart; bottom images (trichrome staining, x20) show fibrosis in the KO heart. Scale bar=40 µm.

Heart Failure Marker Gene Expression in ET-1 KO Mice
To characterize the molecular phenotype of the dilated cardiomyopathy, we evaluated the expression of several genes regulated in other models of heart failure. All showed the same pattern as seen in other heart failure models (Data Supplement Table II). Expression of atrial natriuretic factor, brain natriuretic peptide, and ßMHC expression was increased, and message for MHC, SERCA2a, and phospholamban was less abundant in the ET-1–deleted hearts. Real-time results were obtained in triplicate on each sample of cardiac RNA from which an average expression level was derived. At least 3 hearts were analyzed for each experimental group evaluated, and the mean expression level was compared between groups to obtain the average fold change.

Aortic Banding Accelerated the Development of Heart Failure in ET-1 KO Mice
To observe the role of cardiomyocte ET-1 in response to hemodynamic load, we challenged the mouse hearts with transverse aortic constriction (TAC). Aortic arch constriction to 27-gauge diameter was performed in 2-month-old mice. After they recovered from surgery, the mice were evaluated with echocardiography by an operator blinded to genotype. Strikingly, the ET-1 KO mice developed dilated heart failure in 3 weeks (Data Supplement Table III). LV function of the WT mice remained within the normal range, and LV hypertrophy developed as expected. LV dysfunction progressed in the ET-1–deleted hearts, as did hypertrophy in the nondeleted sibs (Figure 4). Once again, histological study revealed heterogeneity of cell size, cell loss, disorganization of muscle fibers, and increased fibrosis only in ET-1 KO animals (data not shown).

View larger version (23K): [in this window] [in a new window]   Figure 4. TAC accelerated the development of dilated cardiomyopathy in KO animals. Twenty-seven–gauge TAC was performed in 8- to 10-week-old animals. LV function was measured by echocardiography on conscious animals at 3, 5, and 8 weeks after surgery (A–C). D, Hematoxylin and eosin staining of 4-chamber views of representative banded hearts from WT and KO animals.

ET-1 KO Mice Had Increased Apoptosis in Heart
To explore the mechanism of development of dilated cardiomyopathy in ET-1 KO mice, we used a TUNEL assay to compare apoptosis between the hearts of ET-1 KO and WT animals. An example of this analysis is shown in Figure 5B. At the age of 6 months, the ET-1 KO animals had substantially more abundant TUNEL-positive cells in ventricles compared with their normal siblings (Figure 5A) at a time when ventricular function was still preserved. Confirmatory evidence of increased apoptosis in the ET-1 KO hearts was obtained from Western blots that showed greater cleavage of caspase-3 (Figure 5C). At 8 months of age, the ET-1 KO mouse hearts still had increased TUNEL-positive cells but fewer than at 6 months.

View larger version (33K): [in this window] [in a new window]   Figure 5. Apoptosis assays of hearts from 6-month-old animals. After a TUNEL assay, apoptotic cells were counted on 2 four-chamber sections from each heart and show markedly more apoptosis in the KO mice. A, Statistical summary of this assay (n=8 for each; P<0.05). B, Two x40x views of TUNEL assays from WT and KO. Dense yellow spots represent TUNEL-positive nuclei. C, Western blot analyses of important molecules in apoptosis pathways. Western blots were performed on equal amounts of cardiac lysate from individual WT and KO mice. Top left, Caspase-8 and TRAF1, which participate in TNF-mediated apoptosis; top right, APAF-1 and Bax, which are important in mitochondria-mediated apoptosis. The cleaved, active form of caspase-3, which is activated by both pathways, is shown at the bottom. It is more abundant in all of the ET-1 KO mice (-tubulin was used as a loading control). Quantification may be found in the text. Scale bar=40 µm.

FasL/TNF Signaling Pathway Involved in Increased Apoptosis in KO Hearts
We anticipated that the increased susceptibility of the ET KO mice to cardiac apoptosis would be reflected in levels of mRNA and protein for components of apoptotic signaling. We screened key signaling molecules involved in either the TNF/FasL- or the mitochondria-mediated pathways (upstream of caspase-3 activation) by real-time PCR. We found that transcripts encoding modulators of TNF signaling were regulated in such a way to suggest enhanced responsiveness of that pathway. Caspase-8, which activates caspase-3 in the TNF pathway, as well as NF-Bp65 and TRAF1, inhibitors of this pathway, were regulated 1.6-, –30-, and –3.4-fold, respectively, compared with WT controls, in 6-month-old animals. However, the abundance of the transcript for caspase-9, which encodes the principal mediator of caspase-3 activation by the mitochondrial pathway, was similar in both mouse strains. Moreover, both the message and protein abundance of APAF-1 (1.1-fold; P=0.13) and Bax, proteins commonly upregulated in mitochondria-mediated apoptosis, was similar in ET-1 KO and WT animals (Figure 5C), whereas the protein abundance of caspase-8 was higher (1.7-fold; P=0.0002) and TRAF1 was lower (0.6-fold; P=0.01) in the KO hearts. Ultimately, the amount of activated caspase-3 was higher in the KO hearts (2.7-fold; P=0.002). In addition, transmission electron microscopy showed no gross differences between the mitochondria of the KO and WT hearts (data not shown). Although most regulation of apoptosis is thought to occur at a posttranslational level, transcriptional regulation also plays a role²²; this may be particularly important in the chronic processes that we are examining. Overall, these data suggested that increased apoptosis in KO hearts is due predominantly to increased FasL/TNF signaling rather than the mitochondria-mediated pathway.

Decreased NF-B Gene Expression, Protein Phosphorylation, and Translocation in KO Mice
To examine potential connections between ET-1 signaling and FasL/TNF-mediated apoptosis, we explored NF-B as a downstream target of ERK1/2. Previous studies^20,23 suggested that NF-B activity is regulated by ERK1/2. The inhibitory role of NF-B for FasL/TNF-mediated apoptosis has been established in several models.^24,25 We first examined gene regulation of NF-B by real-time PCR and identified a substantial decrease in NF-Bp65 transcript in 6-month-old KO animals compared with age-matched WT mice, as well as slightly diminished expression in 2-month-old KO hearts. Similarly, the total protein level of NF-Bp65 also is lower, albeit less strikingly, in the KO hearts (77±10% of control; P<0.05). To compare the activity of NF-B, we examined the amount of phosphorylated NF-Bp65 by Western blot and NF-B DNA binding activity by EMSA in cardiac nuclear proteins from animals stressed by hyperthyroidism. Both results showed a substantial reduction in NF-B activity in the cardiomyocytes of KO animals (Figure 6).

View larger version (25K): [in this window] [in a new window]   Figure 6. Cardiac ET-1 impinges on NF-B pathway. A, Changes in protein abundance derived from Western blot analyses of KO and WT hearts. Phosphorylated NF-Bp65 was determined in nuclear proteins from 2-month-old hearts after 2 days of thyroid treatment. Comparison is significant at P<0.01 for this lysate. All other results were from 6-month-old whole-heart lysates. Comparisons are significant at P<0.05 for these. B, NF-B binding activity revealed by EMSA using nuclear protein from hearts after 2 days of thyroid treatment. C, Bar graph shows the intensity of the upper retarded band, allowing comparison of NF-B binding activity between WT and KO.

Diminished Expression of NF-B–Regulated Genes
To extend our analysis of the diminished NF-B activity in KO animal hearts, we looked at the expression of NF-B–regulated genes, including cIAP1, cIAP2, IB, and TRAF1. Real-time PCR results showed that all of them were downregulated in 6-month-old KO mice (2.0-, 2.0-, 1.6-, and 3.4-fold). Western blot analyses also showed decreased cIAP1 and IB (71% and 63% of control, respectively).

ET-1 Inhibits TNF-Induced Apoptosis in Cardiomyocytes
To further confirm the effect of ET-1 on TNF signaling pathway, we examined cultured neonatal cardiomyocytes. After 36 hours of serum deprivation and treatment, TNF induces apoptosis of cardiomyocytes. This induction can be inhibited by ET-1. Bosentan, a competitive antagonist of ET-1 receptors, reverses the inhibition of ET-1. We also measured NF-B binding activity by EMSA. ET-1 slightly increased NF-B binding activity in nuclei, and bosentan blocked this effect. TNF substantially increased NF-B binding activity in nuclei; ET-1 further increased this activity; and again, the effect was blocked by bosentan (Figure 7). Similar results were obtained with the pNF-B–SEAP reporter system (compared with control, ET-1 induced a 2.1±0.19-fold increase in SEAP, P<0.05; bosentan brought the amount of SEAP back to 0.87±0.09-fold, P=0.6 versus control, P<0.01 versus ET-1).

View larger version (29K): [in this window] [in a new window]   Figure 7. ET-1 inhibits TNF-induced apoptosis in rat neonatal cardiomyocytes. A–D, Micrographs showing TUNEL-positive nuclei after 36 hours of treatment. After 48 hours of incubation in serum-free media, bosentan (B; 10 µmol/L), ET-1 (100 nmol/L), and TNF (20 µg/mL) were added sequentially with 1 hour between treatments. A, Serum-free media as control; B, ET-1; C, TNF; D, ET-1+TNF. E, Quantification of TUNEL-positive nuclei in these cells. For control vs ET-1, P=0.22; for control vs TNF, P=7.6x10–5; for TNF vs ET-1+TNF, P=0.030; for ET-1 vs B+ET-1, P=0.0037. F, NF-B binding activity in nuclei of cardiomyocytes from these same treatments. For TNF vs ET-1+TNF, P=0.0062; for ET-1+TNF vs B+ET-1+TNF, P=0.019.

Deletion of ET-1 Gene in Mouse Heart Impairs Its Ability to Increase NF-B Binding Activity in Response to Aortic Banding
We further examined the effect of 27-gauge TAC on NF-B binding activity in the hearts of 2-month-old mice. After 2 days of 27-gauge TAC, the cardiac function of both WT and KO mice was unchanged. However, the increase in NF-B binding activity in WT mouse hearts was significantly inhibited in KO mouse hearts (WT versus KO in phosphorimager counts: before TAC, 1.7x10⁵±10% versus 1.5x10⁵±11%, P>0.05; after TAC, 3.7x10⁵±11% versus 2.7x10⁵±3%, P<0.05; n=3 for each).

	Discussion

Top Abstract Introduction Methods Results Discussion Conclusions References

 
In this study, we showed that cardiomyocyte-specific deletion of ET-1 leads to dilated cardiomyopathy either spontaneously with aging or with the extrinsic challenge of aortic banding. We identified increased apoptosis that correlated with the development of cardiomyopathy in these animals and suggested that this apoptosis is likely to be mediated by the FasL/TNF signaling pathway. We went on to support a role for NF-B signaling in connecting the ET-1 axis with FasL/TNF-mediated apoptosis. These results indicate that cardiomyocyte-derived ET-1 is essential for these cells to survive in response to age or hemodynamic stress.

Several previous investigations have shown that ET-1 has direct effects on myocardial cells, including hypertrophy²⁶ and increasing contractility.⁴ Such effects presumably are mediated by the 2 types of Gq-coupled endothelin receptors present on cardiomyocytes, ET_A and ET_B. However, the plasma concentration of endothelin ranges from 10^–12 to 10^–13 mol/L, which is much lower than the concentrations required to obtain physiological effects on cardiomyocytes in cell culture experiments (nanomoles per liter). This also is probably too low to exert an effect on receptors, with a Kd of 20 pmol/L,²⁷ and suggests that ET-1 acts in a paracrine or autocrine fashion in the heart.

Elucidation of such a paracrine role for ET-1 in the heart has been limited. In rat studies, pharmacological blockade of ET receptors has produced benefits in heart failure, but these studies are limited by the inevitable systemic effects of the agents on blood pressure, volume status, and peripheral resistance.²⁸ Conventional deletion of the endothelin gene produces a lethal phenotype, with suffocation at birth resulting from developmental defects in pharyngeal structures.⁹ Cardiac-specific overexpression of ET-1 also has been difficult, and until recently, published reports have been limited to only modest overexpression with negligible phenotype.²⁹ Substantial cardiomyocyte overexpression of ET-1 in the heart recently was reported to produce a dilated cardiomyopathy, but the marked overexpression of a human transgene and inflammatory myocarditis might have contributed to this finding. Concomitant treatment with ET receptor blocking agents only partially improved the phenotype. A cardiac-specific ET_A-deficient model also has been created.³⁰ These mice are viable and exhibit a normal phenotype at 6 weeks, similar to our young ET-1–deficient mice. They also exhibited normal hypertrophic and contractile responses to 10-day infusion of angiotensin II or isoproterenol. Interestingly, they have upregulation of the ET_B receptor, possibly compensating for the reduction in ET_A. One prediction of our findings is that these mice, lacking the putative protective effects of ET-1 signaling, would show increased apoptosis in response to stress if the ET_A receptor is mediating this effect.

Our cardiac ET-1–deficient mice were normal when young. They developed heart failure only with aging or increased afterload. ET-1 normally is upregulated in the heart in response to a variety of stresses, including hyperthyroidism, increased afterload, hormones such as angiotensin II and adrenaline, and various cytokines. In the absence of ET-1 peptide, the hypertrophic and inotropic influences of this molecule would be missing, possibly leading to inadequate compensation to the stress and, for example, greater-than-appropriate dilation, generating increased wall stress and tissue hypoperfusion, with a resulting augmentation of TNF-mediated apoptosis.³¹ It also is possible that the paracrine effects of cardiomyocyte ET-1 on other cell types such as cardiac fibroblasts might affect remodeling of the heart in response to stress.

In our study, the prolonged exposure, over many months, to Cre recombinase might raise the concern that the recombinase itself could exert toxic effects. However, an echocardiogram of a 15-month-old Cre⁺ mouse (lacking loxP sites in the ET-1 gene) showed normal ventricular function (data not shown). The young ET-1 KO mice subjected to aortic banding recapitulate the dilated cardiomyopathy phenotype, but Cre⁺ mice did not in 3 weeks (data not shown). In a recent report,³² using the same MHC-Cre allele, normal ventricular function was observed in a large number of 8-month-old mice.

An antiapoptotic activity of ET-1 has recently been recognized in cancer cells, endothelial cells, human smooth muscle cells, and cardiomyocytes.^7,33–38 As in most tissues, there are 2 principal pathways for apoptosis in cardiomyocytes, the FasL/TNF extrinsic pathway and the mitochondrial-related route. From the degree of regulation seen in components of both pathways, we suggest that ET-1 is exerting an antiapoptotic effect in the heart predominantly through downregulation of the FasL/TNF pathway. Our data do not exclude the involvement of the mitochondrial pathway, which others have suggested is important in cardiomyocyte apoptosis.³⁹ However, the components of this pathway were similarly abundant in both ET-1 KO and WT animals. In contrast, in the FasL/TNF pathway, decreased TRAF1, decreased NF-B, decreased cIAPs, and increased caspase-8 expression all suggest that there is enhanced FasL/TNF receptor pathway activity in 6-month-old ET-1 KO hearts. Several studies have shown that NF-B strongly inhibits the apoptotic activation of TNF.^40,41 We propose that the anti-apoptotic effect of NF-B would be diminished in the ET-1 KO animal in which NF-B activity is downregulated.⁴² Both ET-1 and TNF increase in the stressed heart,^31,43–47 and cardiac-specific expression of TNF has previously been shown to produce dilated cardiomyopathy, presumably through intact TNF-related apoptosis.⁴⁸ In our study, we found that ET-1 can increase NF-B activity in cultured cardiomyocytes and thereby inhibit TNF induced apoptosis. Similarly, we observed a significant increase in NF-B activity in stressed (TAC or T3) WT mouse hearts, but this increase was diminished in KO mouse hearts. Therefore, we conclude that paracrine/autocrine ET-1 signaling is needed to modulate TNF activation and to preserve cardiomyocytes in the face of various stresses and that NF-B is at least one of the connections between these 2 signaling pathways. A schematic of the putative inhibitory role of ET-1 on TNF signaling is presented in Figure 8.

View larger version (23K): [in this window] [in a new window]   Figure 8. Proposed mechanism of myocyte death in ET-1–deficient heart. In the normal heart, ET-1 may act to induce hypertrophy as a response to stress and may reduce the proapoptotic effects of stressful stimuli. In normal cardiomyocytes, several molecules restrain the apoptotic effect of the TNF signaling pathway. When the modulating effect of ET-1 is withdrawn, several of these antiapoptotic factors are downregulated (suggested by a red X), and signaling down the TNF pathway, stimulated by stress, is increased, culminating in apoptosis.

Our observation may have important clinical ramifications. The nonspecific antagonist of ET-1 receptors, bosentan, is already used widely to treat primary pulmonary hypertension. Such treatment is anticipated to be long term, with no obvious limitation on therapeutic course discussed in the literature.⁴⁹ However, on the basis of this study, we suggest that reduced ET-1 signaling may increase the vulnerability of the heart to stress, including the pulmonary hypertension itself. Although we are unaware of evidence for acceleration of right ventricular or LV dysfunction with bosentan treatment, we would recommend that a careful assessment be obtained in patients treated chronically with this drug. Moreover, in several recent studies of endothelin receptor antagonists in heart failure,^13,50 the disappointing results could be due to this increased vulnerability of the cardiomyocyte to apoptosis, despite the likely beneficial effect of such agents on afterload and myocardial oxygen consumption. Perhaps an agent that distributed poorly to myocardium would be more effective, isoform-specific receptor modulation would be more successful, or patients with low levels of TNF would obtain greater benefit.

	Conclusions

Top Abstract Introduction Methods Results Discussion Conclusions References

 
Our study demonstrates that cardiomyocyte ET-1 is crucial in maintaining normal cardiac remodeling and cardiomyocyte survival in response to age and increased afterload. We further show that this cardioprotective effect of local ET-1 may be mediated by inhibition of FasL/TNF signaling–related apoptosis, influenced at least in part by NF-B.

	Acknowledgments

 
We thank Dr Yongli Kong for performing aortic banding surgery. We are grateful to John Shelton and Dr James Richardson for skilled assistance with histology.

Funding

This work was supported by the NIH/National Heart Lung and Blood Institute (HL-64041 and HL-73449) and the Program in Genomic Applications (HL-66880) (R.V.S.).

Disclosures

None.

	References

Top Abstract Introduction Methods Results Discussion Conclusions References

 
1. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, Masaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature. 1988; 332: 411–415.[CrossRef][Medline] [Order article via Infotrieve]

2. Ishikawa T, Yanagisawa M, Kimura S, Goto K, Masaki T. Positive chronotropic effects of endothelin, a novel endothelium-derived vasoconstrictor peptide. Pflugers Arch. 1988; 413: 108–110.[CrossRef][Medline] [Order article via Infotrieve]

3. Ito H, Hirata Y, Hiroe M, Tsujino M, Adachi S, Takamoto T, Nitta M, Taniguchi K, Marumo F. Endothelin-1 induces hypertrophy with enhanced expression of muscle-specific genes in cultured neonatal rat cardiomyocytes. Circ Res. 1991; 69: 209–215.[Abstract/Free Full Text]

4. Ishikawa T, Yanagisawa M, Kimura S, Goto K, Masaki T. Positive inotropic action of novel vasoconstrictor peptide endothelin on guinea pig atria. Am J Physiol. 1988; 255: H970–H973.[Medline] [Order article via Infotrieve]

5. Ito H, Hirata Y, Adachi S, Tanaka M, Tsujino M, Koike A, Nogami A, Murumo F, Hiroe M. Endothelin-1 is an autocrine/paracrine factor in the mechanism of angiotensin II–induced hypertrophy in cultured rat cardiomyocytes. J Clin Invest. 1993; 92: 398–403.[Medline] [Order article via Infotrieve]

6. Kedzierski RM, Yanagisawa M. Endothelin system: the double-edged sword in health and disease. Annu Rev Pharmacol Toxicol. 2001; 41: 851–876.[CrossRef][Medline] [Order article via Infotrieve]

7. Araki M, Hasegawa K, Iwai-Kanai E, Fujita M, Sawamura T, Kakita T, Wada H, Morimoto T, Sasayama S. Endothelin-1 as a protective factor against beta-adrenergic agonist-induced apoptosis in cardiac myocytes. J Am Coll Cardiol. 2000; 36: 1411–1418.[Abstract/Free Full Text]

8. Clouthier DE, Hosoda K, Richardson JA, Williams SC, Yanagisawa H, Kuwaki T, Kumada M, Hammer RE, Yanagisawa M. Cranial and cardiac neural crest defects in endothelin-A receptor-deficient mice. Development. 1998; 125: 813–824.[Abstract]

9. Kurihara Y, Kurihara H, Suzuki H, Kodama T, Maemura K, Nagai R, Oda H, Kuwaki T, Cao WH, Kamada N, Jishage K, Ouchi Y, Azuma S, Toyoda Y, Ishikawa T, Kumada M, Yazaki Y. Elevated blood pressure and craniofacial abnormalities in mice deficient in endothelin-1. Nature. 1994; 368: 703–710.[CrossRef][Medline] [Order article via Infotrieve]

10. Yanagisawa H, Yanagisawa M, Kapur RP, Richardson JA, Williams SC, Clouthier DE, de Wit D, Emoto N, Hammer RE. Dual genetic pathways of endothelin-mediated intercellular signaling revealed by targeted disruption of endothelin converting enzyme-1 gene. Development. 1998; 125: 825–836.[Abstract]

11. Chien KR. To Cre or not to Cre: the next generation of mouse models of human cardiac diseases. Circ Res. 2001; 88: 546–549.[Free Full Text]

12. Sakai S, Miyauchi T, Kobayashi M, Yamaguchi I, Goto K, Sugishita Y. Inhibition of myocardial endothelin pathway improves long-term survival in heart failure. Nature. 1996; 384: 353–355.[CrossRef][Medline] [Order article via Infotrieve]

13. Krum H, Liew D. Current status of endothelin blockade for the treatment of cardiovascular and pulmonary vascular disease. Curr Opin Investig Drugs. 2003; 4: 298–302.[Medline] [Order article via Infotrieve]

14. Shohet RV, Kisanuki YY, Zhao XS, Siddiquee Z, Franco F, Yanagisawa M. Mice with cardiomyocyte-specific disruption of the endothelin-1 gene are resistant to hyperthyroid cardiac hypertrophy. Proc Natl Acad Sci U S A. 2004; 101: 2088–2093.[Abstract/Free Full Text]

15. Hill JA, Rothermel B, Yoo KD, Cabuay B, Demetroulis E, Weiss RM, Kutschke W, Bassel-Duby R, Williams RS. Targeted inhibition of calcineurin in pressure-overload cardiac hypertrophy: preservation of systolic function. J Biol Chem. 2002; 277: 10251–10255.[Abstract/Free Full Text]

16. Yang XP, Liu YH, Rhaleb NE, Kurihara N, Kim HE, Carretero OA. Echocardiographic assessment of cardiac function in conscious and anesthetized mice. Am J Physiol. 1999; 277: H1967–H1974.[Medline] [Order article via Infotrieve]

17. Zhao XS, Gallardo TD, Lin L, Schageman JJ, Shohet RV. Transcriptional mapping and genomic analysis of the cardiac atria and ventricles. Physiol Genomics. 2002; 12: 53–60.[Abstract/Free Full Text]

18. Wang Z, Wang DZ, Hockemeyer D, McAnally J, Nordheim A, Olson EN. Myocardin and ternary complex factors compete for SRF to control smooth muscle gene expression. Nature. 2004; 428: 185–189.[CrossRef][Medline] [Order article via Infotrieve]

19. Jiang B, Haverty M, Brecher P. N-acetyl-L-cysteine enhances interleukin-1beta–induced nitric oxide synthase expression. Hypertension. 1999; 34: 574–579.[Abstract/Free Full Text]

20. Jiang B, Xu S, Hou X, Pimentel DR, Brecher P, Cohen RA. Temporal control of NF-kappaB activation by ERK differentially regulates interleukin-1beta-induced gene expression. J Biol Chem. 2004; 279: 1323–1329.[Abstract/Free Full Text]

21. Molkentin JD, Lu JR, Antos CL, Markham B, Richardson J, Robbins J, Grant SR, Olson EN. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell. 1998; 93: 215–228.[CrossRef][Medline] [Order article via Infotrieve]

22. Kumar S, Cakouros D. Transcriptional control of the core cell-death machinery. Trends Biochem Sci. 2004; 29: 193–199.[CrossRef][Medline] [Order article via Infotrieve]

23. Moon SK, Cha BY, Kim CH. ERK1/2 mediates TNF-alpha-induced matrix metalloproteinase-9 expression in human vascular smooth muscle cells via the regulation of NF-kappaB and AP-1: involvement of the ras dependent pathway. J Cell Physiol. 2004; 198: 417–427.[CrossRef][Medline] [Order article via Infotrieve]

24. Karin M, Lin A. NF-kappaB at the crossroads of life and death. Nat Immunol. 2002; 3: 221–227.[CrossRef][Medline] [Order article via Infotrieve]

25. Misra A, Haudek SB, Knuefermann P, Vallejo JG, Chen ZJ, Michael LH, Sivasubramanian N, Olson EN, Entman ML, Mann DL. Nuclear factor-B protects the adult cardiac myocyte against ischemia-induced apoptosis in a murine model of acute myocardial infarction. Circulation. 2003; 108: 3075–3078.[Abstract/Free Full Text]

26. Sugden PH. An overview of endothelin signaling in the cardiac myocyte. J Mol Cell Cardiol. 2003; 35: 871–886.[CrossRef][Medline] [Order article via Infotrieve]

27. Desmarets J, Gresser O, Guedin D, Frelin C. Interaction of endothelin-1 with cloned bovine ETA receptors: biochemical parameters and functional consequences. Biochemistry. 1996; 35: 14868–14875.[CrossRef][Medline] [Order article via Infotrieve]

28. Ito H, Hiroe M, Hirata Y, Fujisaki H, Adachi S, Akimoto H, Ohta Y, Marumo F. Endothelin ETA receptor antagonist blocks cardiac hypertrophy provoked by hemodynamic overload. Circulation. 1994; 89: 2198–2203.[Abstract/Free Full Text]

29. Yang LL, Gros R, Kabir MG, Sadi A, Gotlieb AI, Husain M, Stewart DJ. Conditional cardiac overexpression of endothelin-1 induces inflammation and dilated cardiomyopathy in mice. Circulation. 2004; 109: 255–261.[Abstract/Free Full Text]

30. Kedzierski RM, Grayburn PA, Kisanuki YY, Williams CS, Hammer RE, Richardson JA, Schneider MD, Yanagisawa M. Cardiomyocyte-specific endothelin A receptor knockout mice have normal cardiac function and an unaltered hypertrophic response to angiotensin II and isoproterenol. Mol Cell Biol. 2003; 23: 8226–8232.[Abstract/Free Full Text]

31. Paulus WJ. Cytokines and heart failure. Heart Fail Monit. 2000; 1: 50–56.[Medline] [Order article via Infotrieve]

32. Jacoby JJ, Kalinowski A, Liu MG, Zhang SS, Gao Q, Chai GX, Ji L, Iwamoto Y, Li E, Schneider M, Russell KS, Fu XY. Cardiomyocyte-restricted knockout of STAT3 results in higher sensitivity to inflammation, cardiac fibrosis, and heart failure with advanced age. Proc Natl Acad Sci U S A. 2003; 100: 12929–12934.[Abstract/Free Full Text]

33. Ogata Y, Takahashi M, Ueno S, Takeuchi K, Okada T, Mano H, Ookawara S, Ozawa K, Berk BC, Ikeda U, Shimada K, Kobayashi E. Antiapoptotic effect of endothelin-1 in rat cardiomyocytes in vitro. Hypertension. 2003; 41: 1156–1163.[Abstract/Free Full Text]

34. Lassiter LK, Carducci MA. Endothelin receptor antagonists in the treatment of prostate cancer. Semin Oncol. 2003; 30: 678–688.[CrossRef][Medline] [Order article via Infotrieve]

35. Rosano L, Spinella F, Salani D, Di Castro V, Venuti A, Nicotra MR, Natali PG, Bagnato A. Therapeutic targeting of the endothelin a receptor in human ovarian carcinoma. Cancer Res. 2003; 63: 2447–2453.[Abstract/Free Full Text]

36. Peduto Eberl L, Bovey R, Juillerat-Jeanneret L. Endothelin-receptor antagonists are proapoptotic and antiproliferative in human colon cancer cells. Br J Cancer. 2003; 88: 788–795.[CrossRef][Medline] [Order article via Infotrieve]

37. Eberl LP, Egidy G, Pinet F, Juillerat-Jeanneret L. Endothelin receptor blockade potentiates FasL-induced apoptosis in colon carcinoma cells via the protein kinase C-pathway. J Cardiovasc Pharmacol. 2000; 36: S354–S356.[Medline] [Order article via Infotrieve]

38. Shichiri M, Yokokura M, Marumo F, Hirata Y. Endothelin-1 inhibits apoptosis of vascular smooth muscle cells induced by nitric oxide and serum deprivation via MAP kinase pathway. Arterioscler Thromb Vasc Biol. 2000; 20: 989–997.[Abstract/Free Full Text]

39. Adams JW, Brown JH. G-proteins in growth and apoptosis: lessons from the heart. Oncogene. 2001; 20: 1626–1634.[CrossRef][Medline] [Order article via Infotrieve]

40. Bergmann MW, Loser P, Dietz R, von Harsdorf R. Effect of NF-kappa B Inhibition on TNF-alpha-induced apoptosis and downstream pathways in cardiomyocytes. J Mol Cell Cardiol. 2001; 33: 1223–1232.[CrossRef][Medline] [Order article via Infotrieve]

41. Levkau B, Scatena M, Giachelli CM, Ross R, Raines EW. Apoptosis overrides survival signals through a caspase-mediated dominant-negative NF-kappa B loop. Nat Cell Biol. 1999; 1: 227–233.[CrossRef][Medline] [Order article via Infotrieve]

42. Mangelus M, Galron R, Naor Z, Sokolovsky M. Involvement of nuclear factor-kappaB in endothelin-A-receptor-induced proliferation and inhibition of apoptosis. Cell Mol Neurobiol. 2001; 21: 657–674.[CrossRef][Medline] [Order article via Infotrieve]

43. Csiszar A, Ungvari Z, Koller A, Edwards JG, Kaley G. Aging-induced proinflammatory shift in cytokine expression profile in coronary arteries. FASEB J. 2003; 17: 1183–1185.[Abstract/Free Full Text]

44. Mann DL. Stress-activated cytokines and the heart: from adaptation to maladaptation. Annu Rev Physiol. 2003; 65: 81–101.[CrossRef][Medline] [Order article via Infotrieve]

45. Diwan A, Tran T, Misra A, Mann DL. Inflammatory mediators and the failing heart: a translational approach. Curr Mol Med. 2003; 3: 161–182.[CrossRef][Medline] [Order article via Infotrieve]

46. Baumgarten G, Knuefermann P, Kalra D, Gao F, Taffet GE, Michael L, Blackshear PJ, Carballo E, Sivasubramanian N, Mann DL. Load-dependent and -independent regulation of proinflammatory cytokine and cytokine receptor gene expression in the adult mammalian heart. Circulation. 2002; 105: 2192–2197.[Abstract/Free Full Text]

47. Palmieri EA, Benincasa G, Di Rella F, Casaburi C, Monti MG, De Simone G, Chiariotti L, Palombini L, Bruni CB, Sacca L, Cittadini A. Differential expression of TNF-alpha, IL-6, and IGF-1 by graded mechanical stress in normal rat myocardium. Am J Physiol Heart Circ Physiol. 2002; 282: H926–H934.[Abstract/Free Full Text]

48. Bryant D, Becker L, Richardson J, Shelton J, Franco F, Peshock R, Thompson M, Giroir B. Cardiac failure in transgenic mice with myocardial expression of tumor necrosis factor-alpha. Circulation. 1998; 97: 1375–1381.[Abstract/Free Full Text]

49. Rubin LJ, Badesch DB, Barst RJ, Galie N, Black CM, Keogh A, Pulido T, Frost A, Roux S, Leconte I, Landzberg M, Simonneau G. Bosentan therapy for pulmonary arterial hypertension. N Engl J Med. 2002; 346: 896–903.[Abstract/Free Full Text]

50. Rich S, McLaughlin VV. Endothelin receptor blockers in cardiovascular disease. Circulation. 2003; 108: 2184–2190.[Abstract/Free Full Text]

---

 

CLINICAL PERSPECTIVE

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide secreted by endothelium. It also is expressed in the heart and has direct influence on cardiac myocytes, including hypertrophic and inotropic effects. Despite a favorable hemodynamic result from inhibition of ET-1 signaling, pharmacological inhibitors of ET receptors have not performed well in trials for congestive heart failure but are used for treatment of pulmonary hypertension. To study the local cardiac effects of ET-1, we created a transgenic mouse with cardiomyocyte-specific deletion of the ET-1 gene. These mice develop a dilated cardiomyopathy as they age or in response to the stress of aortic banding. We show that ET-1 appears to exert an antiapoptotic effect by stimulating nuclear factor-B inhibition of tumor necrosis factor–mediated apoptosis. Thus, endothelin may be important for cardiomyocyte survival under stress. This implies a potential proapoptotic effect of ET receptor blockers that could explain their disappointing results in heart failure trials. It also may suggest caution in the long-term use of ET-1 blockade. Ultimately, a deeper understanding of ET-1 action should point to more refined and effective therapies, perhaps relating to receptor-specific inhibition or agents with less avidity for cardiac tissue.

	Footnotes

 
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.105.577288/DC1.

This article has been cited by other articles:

				M Borjesson and A Pelliccia Incidence and aetiology of sudden cardiac death in young athletes: an international perspective Br. J. Sports Med., September 1, 2009; 43(9): 644 - 648. [Abstract] [Full Text] [PDF]

				R M Campbell, S Berger, and J Drezner Sudden cardiac arrest in children and young athletes: the importance of a detailed personal and family history in the pre-participation evaluation Br. J. Sports Med., May 1, 2009; 43(5): 336 - 341. [Abstract] [Full Text] [PDF]

				J. D. Rothmier and J. A. Drezner The Role of Automated External Defibrillators in Athletics Sports Health: A Multidisciplinary Approach, January 1, 2009; 1(1): 16 - 20. [Abstract] [Full Text] [PDF]

				J. A Drezner and K. Khan Sudden cardiac death in young athletes BMJ, July 3, 2008; 337(jul03_2): a309 - a309. [Full Text]

				C.-C. Juan, T.-Y. Chuang, C.-C. Lien, Y.-J. Lin, S.-W. Huang, C. F. Kwok, and L.-T. Ho Leptin increases endothelin type A receptor levels in vascular smooth muscle cells Am J Physiol Endocrinol Metab, March 1, 2008; 294(3): E481 - E487. [Abstract] [Full Text] [PDF]

				M. Kang, K. Y. Chung, and J. W. Walker G-Protein Coupled Receptor Signaling in Myocardium: Not for the Faint of Heart Physiology, June 1, 2007; 22(3): 174 - 184. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 114/8/830    most recent CIRCULATIONAHA.105.577288v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhao, X.-S. Articles by Shohet, R. V. Search for Related Content PubMed PubMed Citation Articles by Zhao, X.-S. Articles by Shohet, R. V. Related Collections Apoptosis Cell signalling/signal transduction Genetically altered mice