We compared the ability of angiotensin II (Ang II) to induce hypertrophy of neonatal rat ventricular myocytes with that of endothelin-1. Over 72 hours, Ang II (1 μmol/L) increased the ratio of protein to DNA by less than 10%, whereas endothelin-1 (100 nmol/L) produced a 28% increase. The growth effects of either agonist occurred independently of chronotropic actions. Radioligand binding studies showed that myocytes have nearly 300-fold more receptors for endothelin-1 than Ang II, and type 1 and type 2 Ang II receptor subtypes (AT1 and AT2) are present in near equal proportions. Cotreatment with a 10-fold molar excess of AT2 antagonists (PD 123177 or CGP 42112) for 72 hours augmented the Ang II–induced increase in the protein-to-DNA ratio to levels nearly as high (23%) as those with endothelin-1 (28%). AT2 antagonists enhanced Ang II stimulation of protein synthesis, as indexed by [3H]leucine incorporation, whereas an AT1 antagonist blocked Ang II–induced incorporation. An AT2 antagonist also prevented Ang II–induced protein degradation. In conclusion, Ang II–induced myocyte growth is tempered because of low AT1 levels and an antigrowth effect of AT2. These findings have potential clinical significance in that regression of hypertension-induced cardiac hypertrophy by AT1 antagonists may be in part due to an unopposed antigrowth effect of Ang II mediated via AT2.
Angiotensin II (Ang II) has been implicated in the cardiac hypertrophy associated with hemodynamic overload, myocardial infarction, and hypertension.1 The central importance of Ang II as a growth factor for the heart was first suggested by the ability of angiotensin-converting enzyme inhibitors to cause regression of cardiac hypertrophy in a variety of pathological conditions.1 Subsequent in vivo studies in animals indicated that Ang II may have direct growth-promoting effects on myocytes independent of effects on hemodynamic afterload.2 3 4 5 These studies have been criticized on several grounds, including differential effects of angiotensin-converting enzyme inhibitors, vasodilators, and sympatholytic agents on heart rate, stroke volume, and diastolic function; the selectivity of angiotensin-converting enzyme inhibitors; the effect of angiotensin-converting enzyme inhibitors on bradykinin levels; and the difficulty of relating hemodynamic changes in a small mammal to humans. Evidence that Ang II is a growth factor for myocytes was first found with cultured embryonic chick myocytes.6 Whether Ang II is capable of inducing hypertrophy of mammalian myocytes, however, is controversial.7 8 9
The NRVM is a useful model system for the study of signal transduction events associated with cardiac hypertrophy. Stimulation of NRVMs by stretch, neurohumoral agents, or phorbol esters that activate PKC induces many of the features of cardiac hypertrophy observed in vivo.10 NRVMs possess components of a local renin-angiotensin system,11 and Ang II can be detected in the conditioned medium of NRVMs, the levels of which are increased by mechanical stretch.12 13 Recently, the autocrine secretion of Ang II was implicated in the hypertrophic response of NRVMs to mechanical stretch.13
Binding studies on membranes prepared from rat heart have indicated that myocytes possess both Ang II receptor subtypes, AT1 and AT2, and that Ang II receptor expression is increased during the neonatal period and decreases with maturation.14 15 This conclusion was corroborated by a recent radioligand binding study on cultured NRVMs.16 AT1 and AT2 receptors can be distinguished from one another on the basis of their affinity for nonpeptide receptor antagonists, such as losartan (AT1) and PD 123177 (AT2). AT1 is responsible for the majority of the effects of Ang II on the heart, including stimulated increases in heart rate, contractility, and growth.1 Little is known about the role of AT2 in the heart or which second messenger systems it activates. Recently, studies on coronary endothelial cells indicated that AT2 is coupled in these cells to an antigrowth process that counteracts the growth-promoting program initiated by AT1 activation.17
The objective of the present study was to critically assess the growth-promoting actions of Ang II on NRVMs and, in doing so, to test the following hypotheses: (1) The reported poor ability of Ang II to induce cellular hypertrophy of NRVMs reflects the low number of membrane receptors, and (2) activation of AT2 counteracts the growth-promoting effects of Ang II that are mediated by AT1 in these cells. The growth-promoting actions of Ang II were compared with those of PMA and ET-1. PMA is a potent activator of PKC and inducer of hypertrophy of NRVMs.18 ET-1 was selected for several reasons: (1) It has been reported to induce hypertrophy of NRVMs19 ; (2) both Ang II and ET-1 have been reported to activate the same signal transduction pathways in cardiomyocytes as well as other cell types, including the activation of phospholipase C, PKC, the MAPK cascade, and tyrosine kinases20 ; and (3) AT1 and endothelin receptors (ETA and ETB) belong to the same superfamily of G protein–coupled receptors with seven transmembrane–spanning domains.21 22
Tissue Culture and Media
Ventricular myocytes were isolated from Sprague-Dawley rat pups as described.23 Myocytes were resuspended in medium containing 32 μg/mL 5-bromo-2′-deoxyuridine (BrdU) and plated at a high density (156 000 cells per centimeter squared) onto plates precoated with 1 g/L gelatin. By using differential plating, adding BrdU to the medium, and plating at a high cell density, we routinely obtained cultures of greater than 90% ventricular myocytes. After 22 hours, the dispersion medium was replaced with serum-free minimum essential medium–serum substitute 223 with (per liter) 1.95 mg cholesterol, 3.2 μg hydrocortisone, 31 mg BrdU, 50 mg ascorbic acid, 9 mg choline chloride, 2 mg protamine sulfate, 0.02 mg vitamin B12, 0.02 mg biotin, 0.72 mg Fe(NO3)3, 0.28 mg ZnSO4·7H2O, 0.01 mg NaSeO4, 660 mg creatine, 396 mg carnitine, and 626 mg taurine. Dosing of cells for growth studies was begun 44 hours later.
Radioligand Binding Studies
Radiolabeled Ang II and ET-1 binding was studied in living cells. Surface (receptor)–bound ligand was distinguished from internalized ligand by acid washing of the cells, a procedure other researchers have used to study Ang II and ET-1 binding.24 25 For Ang II, the binding medium was as described.24 For ET-1, the binding medium was minimum essential medium with 50 mmol/L HEPES (pH 7.4), 0.1 mg/mL bacitracin, 0.1 μg/mL aprotinin, 0.48 μg/mL leupeptin, 0.68 μg/mL pepstatin A, 0.2 mmol/L phenylmethylsulfonyl fluoride, and 2.5 g/L bovine serum albumin. Cells were washed three times with 1.0 mL binding buffer. Studies for determination of the time course of binding were started by addition of 0.17 nmol/L 125I–[Tyr4]-Ang II, 0.05nmol/L 125I–[Tyr4,Sar1,Ile8]-Ang II, or 0.06 nmol/L 125I–[Tyr13]-ET-1 (0.06 to 0.2 μCi/mL) to each plate or well. Incubations were done at 22°C and terminated by aspiration of the binding medium and quick washing of the cells three times with 2.0 mL ice-cold binding medium. Surface-bound radioactivity and internalized radioactivity were determined as described.24 Counts were corrected for nonspecific binding or uptake by subtraction of the radioactivity measured in the presence of excess unlabeled ligand.
Measurement of Cardiac Hypertrophy
The ratio of protein to DNA was used as a measure of cardiomyocyte growth.18 Rates of protein synthesis were assessed by pulse-labeling of cells with [3H]leucine and measurement of its incorporation into acid-precipitable protein. For each tissue dispersion, three 35-mm plates of cells were used per experimental condition. Four hours before the end of a 48-hour incubation, 1.0 μCi/mL [3H]leucine was added to the culture medium. At the end of the incubation, protein was precipitated and radioactivity measured as described.23
For protein degradation studies, cells were pulse-labeled with 1.0 μCi/mL [3H]leucine for 4 hours. Then cells were washed three times with culture medium and either processed for determination of initial (zero time) levels of leucine incorporation or treated with agonist for 48 hours. The amount of [3H]leucine incorporated into protein was measured as described.23 Three 35-mm plates of cells were used per experimental condition, and results were normalized to the amount of protein.
Three protocols were used for assessment of the growth-promoting effects of Ang II. In Protocol A, cells were treated with agonists 68 hours after plating, at which time they had spread out, reaching confluence, and were beating in unison; in protocol B, cells were contraction-arrested with 50 mmol/L KCl for the final 44 hours of the 68 hours preceding plating but were released from arrest at the time of dosing; and in protocol C, cells were contraction-arrested for 44 hours before and during dosing initiated 68 hours after plating. NRVMs undergo rapid growth over the first 5 days in culture,18 and contraction arrest slows basal growth, as reflected in a 15.2±4.2% (n=3) lower protein-DNA ratio present 48 hours after release from arrest. Protocol B offers the following advantages: (1) Basal growth is slowed; (2) sufficient time is provided for recovery from the dispersion, yet cells can be studied within a reasonable time frame; and (3) cells will begin to adapt the muscle phenotype when released from arrest.
Peptide Degradation Studies
The stability of Ang II in the culture medium was determined (1) by radioimmunoassay, using Ang II antiserum (Amersham) and following the manufacturer's recommended protocol, and (2) by use of a bioassay.12 ET-1 breakdown was measured with the Biotrak ELISA system (Amersham).
Ang II antagonist [AT2 peptide: nicotinoyl-Tyr-Lys(Z-Arg)-His-Pro-Ile-OH] was from BACHEM Bioscience. PD 123177 was provided by Dr Joan Keiser of Parke-Davis Pharmaceutical Research. Losartan and EXP 3174 were supplied by DuPont Merck Pharmaceutical Co. l-[4,5-3H(N)]Leucine, 125I–[Tyr4]-Ang II, 125I–[Tyr4,Sar1,Ile8]-Ang II, and 125I–[Tyr13]-ET-1 were from DuPont-NEN. BQ-485 was from Calbiochem-Novabiochem International. Ang II (human) and [Sar1]-Ang II were from Peninsula Laboratories. ET-1 (human and porcine), PMA, and pepstatin A were from Sigma Chemical Co.
Results are reported as mean±SE for n number of determinations on separate tissue dispersions. Statistical significance was determined by either paired Student's t test or one-way ANOVA, followed by the Student-Newman-Keuls test. Because of variability in responsiveness among different dispersions, the data in Fig 5⇓ were analyzed by repeated measures ANOVA.
Ang II Binding
NRVMs have surface binding sites for Ang II, as determined with either 125I–[Tyr4]-Ang II or 125I–[Tyr4,Sar1,Ile8]-Ang II, a protease-resistant Ang II analogue (Fig 1⇓). Ang II and [Sar1,Ile8]-Ang II were both slowly internalized (Fig 1A⇓). With a value of 1 nmol/L for the Kd of Ang II for AT1 and AT2,26 Bmax was calculated from the level of surface binding observed at equilibrium27 and determined to be 36.7±17.2 fmol/mg protein (n=6; ranging from 5.5 to 110.1 fmol/mg protein), or 4500 sites per cell. On the basis of the displacement of surface-bound 125I–[Tyr4]-Ang II by 1 μmol/L losartan or PD 123177, the percentages of AT1 and AT2 sites were found to be 50.1±6.4% and 34.0±6.2% (n=3), respectively, in agreement with other studies.14 15 16
NRVMs show greater binding and internalization of ET-1 than of Ang II (Fig 2⇓). Steady-state levels of surface binding were reached at 1 hour. Levels of internalized ligand continued to increase over 2 hours. Competition binding studies showed that cardiomyocytes have two binding sites for ET-1: 13.4±2.4% high-affinity and 86.6±2.5% low-affinity sites, with IC50 values of 0.021±0.009 and 14.0±2.5 nmol/L (n=3), respectively (Fig 3⇓). From Scatchard analysis, Bmax was determined to be 10.5±0.93 pmol/mg protein, or 1.2×106 sites per cell. The ETA-selective antagonist BQ-485 displaced all specific surface binding of 0.02 nmol/L 125I–[Tyr13]-ET-1, with an IC50 of 4.5±1.8 nmol/L (n=3).
The binding studies indicated that the high- and low-affinity binding sites for ET-1 are 38 and 248 times, respectively, more prevalent than Ang II sites. However, the disparity in the number of binding sites for Ang II and ET-1 may not translate into a similar difference in ability to activate a signal transduction event. This point can be seen if the peak inductions of MAPK activity23 produced by maximal concentrations of Ang II or ET-1 are compared. MAPK activity after a 2-minute exposure to 1 μmol/L Ang II and 100 nmol/L ET-1 was 2.72±0.24 and 6.9±0.35 (n=12) times basal levels, respectively, only a 2.5-fold difference.
Ang II–Induced Hypertrophy of Cardiac Myocytes
Treatment with 1 μmol/L Ang II had a modest effect on protein-DNA ratio, regardless of the protocol (Fig 4⇓). When basal growth was slowed before treatment, 1 μmol/L Ang II increased protein-DNA ratio over 72 hours by 8.9±1.9% (n=9, P<.01). Lower concentrations of Ang II (10 or 100 nmol/L) were not effective in producing consistent increases in protein-DNA ratio. ET-1 (100 nmol/L) and PMA (100 nmol/L) were more effective than Ang II in increasing protein-DNA ratio, particularly when basal rates of growth were slowed (Fig 4B and 4C⇓⇓). With ET-1 and PMA, marked increases in protein-DNA ratio were seen 72 hours after release from contraction arrest (Fig 4B⇓). The growth-promoting effects of ET-1 and PMA were not simply due to a chronotropic action (see below), as both were effective in inducing growth of contraction-arrested cells (Fig 4C⇓). With all three agents, modest increases over control in DNA content per plate were noted after 72 hours (Ang II, 7.2±2.5%; ET-1, 13.7±2.7%; and PMA, 11.5±2.0%, n=4). These increases may represent better cell adhesion and/or a slight slippage of cardiomyocytes through the BrdU block, as NRVMs may undergo limited DNA synthesis.28 Increases over control in protein per plate paralleled the changes in protein-DNA ratio (Ang II, 17.2±4.9%; ET-1, 46.2±10.8%; and PMA, 60.4±6.2%, n=4).
The differing abilities of Ang II and ET-1 to induce hypertrophy were apparently not related to the metabolism of the two peptides. When culture medium was analyzed by radioimmunoassay with an antiserum that recognizes both Ang II and Ang III, agonists with equal affinity for AT1, an appreciable amount (54.2±5.5%, n=3) of agonist was present 24 hours after dosing with 1 μmol/L Ang II. As determined by bioassay,12 the concentration of Ang II/Ang III present after 24 hours was 50.1±25.9% (n=3) of the initial concentration. The observation of an appreciable concentration of agonist after 24 hours is consistent with the finding that daily dosing with Ang II was no more effective in inducing growth than the dosing regimen used. With ET-1, 79.6±0.4% (n=3) of the initial concentration of 100 nmol/L was present after 24 hours.
AT2 Ligands Augment Ang II–Induced Hypertrophy of Cardiac Myocytes
Treatment of cardiomyocytes for 72 hours with either of two AT2 antagonists, AT2 peptide or PD 123177, tended to result in higher protein-DNA ratios (Fig 5⇓), although this effect did not reach statistical significance for either compound. AT2 peptide and PD 123177 are structurally dissimilar compounds that have been reported to show much greater selectivity for AT2 than for AT1.26 AT2 peptide is a markedly modified pentapeptide analogue of Ang II identical to CGP 42112 (CIBA-Geigy). PD 123177 is a tetrahydroimidazo-pyridine derivative. When either was added with Ang II, statistically significant increases in protein-DNA ratio were observed, nearly equivalent to those produced by ET-1 (Figs 4 and 5⇑⇓). In three of five experiments, the increase in protein-DNA ratio with Ang II and AT2 peptide was greater than additive of their individual effects. For PD 123177, this was so in three of four experiments. AT2 peptide and PD 123177 did not have an effect on DNA levels themselves, nor did they affect the modest increases induced by Ang II.
Twenty-four hours after release from arrest, NRVMs beat at a rate of 23.4±4.8 (n=5) per minute. Factors contributing to beating frequency include cell density, how spread out individual cells are, and how much contact there is between cells. The latter two factors are influenced by the amount of time in culture. Ang II modestly stimulated beating after 24 hours by 22.4±5.3% (n=5). No further enhancement of beating was observed with AT2 peptide or PD 123177. Treatment with ET-1 or PMA resulted in a beating frequency (contractions per minute) after 24 hours of 109.8±11.2 and 60.6±17.7 (n=5), respectively.
Role of Protein Synthesis and Degradation
Ang II (1 μmol/L) had a modest effect, ie, 10.2±1.7% increase over control (P<.05), on protein synthesis, as indexed by [3H]leucine incorporation. Neither AT2 peptide nor PD 123177 by itself had any significant effect on leucine incorporation into protein (AT2 peptide, −4.9±3.4%, n=4; PD 123177, −9.6±2.1%, n=5). Cotreatment with Ang II and either AT2 peptide or PD 123177, however, resulted in higher rates of leucine incorporation than with Ang II alone (Fig 6⇓). At a 10-fold molar excess, the AT1 nonpeptide antagonist losartan blocked Ang II–induced leucine incorporation (4.3±2.2% increase over control, n=4).
Over 48 hours, NRVMs exhibited a 58.3±8.3% (n=5) decrease in [3H]leucine incorporated into protein, a measure of protein degradation. Ang II (1 μmol/L) produced a 41.6±21.3% (n=5, P<.05) further loss of incorporated [3H]leucine. With 10 μmol/L AT2 peptide, no significant further decrease (6.1±1.2%, n=3) was observed with Ang II.
Our results show that Ang II has a modest anabolic effect on NRVMs compared with ET-1 or PMA. The lesser growth-promoting action of Ang II on NRVMs was partly due to a low number of AT1 receptors. In addition, we have presented evidence that Ang II exerts an antigrowth effect that can be blocked by AT2 antagonists.
In vivo studies on animals have implicated Ang II as a hypertrophic agent for the heart, independent of effects on hemodynamic afterload.1 2 3 4 5 The growth-promoting actions of Ang II on myocytes could be direct or secondary to increased synthesis and release of other growth factors by either myocytes or nonmyocytes. Evidence that Ang II does act on myocytes to promote growth, directly or through the mediation of other factors, was first reported for embryonic chick myocytes.6 Whether the same scenario applies to mammalian cardiomyocytes is controversial. Using NRVMs, different investigators have reported respectable (25% increases in protein-DNA ratio), modest (25% increases in rates of protein synthesis), or no (as indexed by an increase in cell area) effects of Ang II on growth.7 8 9 Freshly isolated adult rat cardiocytes were reported not to respond to Ang II with an increase in the rate of protein synthesis.29 Our results support the conclusion that Ang II is a weak promoter of NRVM growth, inducing less than a 10% increase in protein-DNA ratio and a 10% increase in protein synthesis. This was the case regardless of whether the chronotropic action of Ang II was blocked. Our conclusion that Ang II was a poor promoter of NRVM growth is at odds with the report by Miyata and Haneda8 of a 25% increase in protein-DNA ratio with 1 μmol/L Ang II after 72 hours but not earlier. However, the pattern of change they reported for protein-DNA ratio over time in both control and Ang II–treated cultures suggests that the Ang II effect at 72 hours was cumulative and partly due to enhancement of marked basal growth.
A major determinant of the ability of Ang II to induce hypertrophy of cultured NRVMs appears to be the low number of AT1 receptors. The calculated Bmax for AT1 on NRVMs is low (37 fmol/mg protein) compared with the number of AT1 receptors present on adult rat hepatocytes (8000 fmol/mg membrane protein, see Reference 30) and neonatal rat cardiac fibroblasts (778 fmol/mg protein, see Reference 31) or ET-1 binding sites (10 500 fmol/mg protein) on NRVMs. Ang II receptor expression in the ventricles is decreased markedly after birth and with maturation.14 15 Other researchers have reported that NRVMs from Wistar rats have 10 times the number of Ang II receptors reported here, although high variability was noted.16 However, this study16 did not distinguish between total uptake and surface-bound Ang II, which would have overestimated binding sites.
One caveat needs to be considered regarding the conclusion that low receptor number predicts a poor physiological response: that is, the coupling efficiency of a receptor to the activation of signal transduction may itself be highly variable. We observed that the difference between the abilities of Ang II and ET-1 to activate MAPK in NRVMs was not nearly as great as predicted based on the difference in receptor number. AT1 may be more tightly coupled than ETA to the activation of signaling pathways.
Cotreatment of NRVMs with Ang II and an AT2 antagonist resulted in increases in protein-DNA ratio that were nearly equal to those seen with ET-1. The growth-promoting effects of the AT2 antagonist were partially due to enhanced protein synthesis. In addition, an AT2 antagonist prevented Ang II–induced protein degradation. These observations are consistent with the hypothesis that AT2 has antigrowth actions in NRVMs. Mounting evidence suggests that AT2 has antigrowth effects in other cells as well: for instance, AT2 is upregulated in rat ovarian follicles with atresia32 ; AT2 has been linked to an antimitotic process in coronary endothelial cells17 ; and transfected AT2 reduced proliferation and inhibited MAPK activity in cultured smooth muscle cells.33
By themselves, the AT2 antagonists tended to produce an increase in protein-DNA ratio. This effect may have resulted from a block in the actions of endogenous Ang II on AT2, levels of which are highly variable. If so, then the failure of the AT2 antagonists to stimulate protein synthesis or block protein degradation on their own may be attributed to limitations in the sensitivity of the experimental methods used as well as the variability of this effect.
A high concentration of Ang II was found to be necessary to produce consistent increases in protein-DNA ratio, reflecting perhaps the importance of receptor recycling rate in the activation of the process coupled to growth. The requirement of a high concentration mandated the use of AT2 ligands at a concentration above their reported IC50 for AT2 but still below their Ki for AT1.26 However, AT2 peptide and PD 123177 are surmountable antagonists, and it is unlikely that they would block Ang II binding to AT1 at a 10-fold molar excess. In fact, they failed to block Ang II–induced leucine incorporation at a 10-fold molar excess in the present study. Nevertheless, we cannot rule out the possibility that these compounds affected Ang II–induced hypertrophy as the result of some allosteric modulation of AT1. A definitive answer to this question will be forthcoming if AT2 levels in NRVMs are dramatically increased via transfection experiments, which are under way. Finally, a nonspecific effect of AT2 peptide or PD 123177 seems unlikely: The compounds are structurally dissimilar, yet with Ang II, higher protein-DNA ratios were seen with both; and losartan and PD 123177 are structurally similar, yet losartan blocked Ang II–induced increases in protein.
In conclusion, we have shown that Ang II is a modest inducer of hypertrophy of NRVMs, reflecting the fact that these cells have a low number of AT1 receptors. In contrast, NRVMs are rich in the ETA receptor, which is a potent inducer of cellular hypertrophy. The effects of Ang II on cell size were enhanced by cotreatment with AT2 antagonists. This effect was due to an enhancement of Ang II–induced rates of protein synthesis as well as an inhibition of Ang II–induced protein degradation.
Selected Abbreviations and Acronyms
|Ang II, III||=||angiotensin II, III|
|AT1, AT2||=||angiotensin receptor type 1, type 2|
|MAPK||=||mitogen-activated protein kinase|
|NRVM||=||neonatal rat ventricular myocyte|
|PKC||=||protein kinase C|
|PMA||=||phorbol 12-myristate 13-acetate|
This work was supported by grants from the National Heart, Lung, and Blood Institute (HL-44883, Dr Baker) and the American Heart Association, Pennsylvania Affiliate (Dr Booz), and by the Geisinger Clinic Foundation. Dr Baker is an Established Investigator of the American Heart Association. The authors thank Lois Carl for her advice on performing the heart dispersions.
Reprint request to George W. Booz, PhD, Weis Center for Research, 26-11, 100 N Academy Ave, Danville, PA 17822. E-mail email@example.com.
- Received September 19, 1995.
- Revision received November 29, 1995.
- Accepted May 17, 1996.
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