Atrial Natriuretic Peptide Induces Apoptosis in Neonatal Rat Cardiac Myocytes*

(Received for publication, December 16, 1996, and in revised form, March 19, 1997)

Can-Fang Wu Dagger §, Nanette H. Bishopric Dagger par and Richard E. Pratt Dagger **

From the Dagger  Falk Cardiovascular Research Center, Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, California 94305 and the  Molecular Cardiology Laboratory, Stanford Research Institute International, Menlo Park, California 94025

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Early heart failure is characterized by elevated plasma atrial natriuretic peptide (ANP) levels, but little is known about the direct effects of ANP on cardiac myocytes. In neonatal rat cardiac myocytes, ANP induced apoptosis in a dose-dependent and cell type-specific manner. Maximum effects occurred at 1 µM ANP, with a 4-5-fold increase in apoptotic cells, reaching a maximum apoptotic index of 19%. In contrast, the maximum apoptotic index of ANP-treated non-myocytes was 1.1 ± 0.2%, equivalent to control cultures. ANP treatment also sharply reduced levels of Mcl-1 mRNA, a Bcl-2 homologue, coincident with the increase in the incidence of apoptosis. ANP induction of apoptosis was receptor-dependent and mediated by cyclic GMP: the effect was mimicked by 8-bromo-cGMP, a membrane-permeable analog, and by sodium nitroprusside, an activator of soluble guanylyl cyclase, and was potentiated by a cGMP-specific phosphodiesterase inhibitor, zaprinast. Interestingly, norepinephrine, a myocyte growth factor, inhibited ANP-induced apoptosis via activation of the beta -adrenergic receptor and elevation of cyclic AMP. These results show that ANP is a specific effector of cardiac myocyte apoptosis in culture via receptor-mediated elevation of cGMP. Furthermore, at least in this model, ANP and norepinephrine may have opposing roles in the modulation of cardiac myocyte growth and survival.


INTRODUCTION

Apoptosis, or programmed cell death, plays a central role in development and homeostasis in all species. Apoptosis differs from necrosis in that it occurs by activation of an energy-requiring molecular suicide program, while necrosis is generally regarded as an accidental or unregulated phenomenon. Apoptosis is seen in cells undergoing embryonic deletion, after serious damage, or following an inappropriate or incomplete growth stimuli. Apoptosis is an important component of organ remodeling in development and during adaptation to stress (1). Both proliferating and quiescent cells are capable of undergoing apoptosis in response to noxious or sublethal events. The pathological activation of apoptosis is now thought to contribute to a variety of disease processes, including heart failure, myocardial infarction, and arrhythmogenic right ventricular dysplasia (2-14). However, the molecular events leading to myocardial apoptosis are poorly understood.

In heart failure, contractile abnormalities are accompanied by multiple compensatory disturbances in myocardial bioenergetics, neural and hormonal circulatory control, and peripheral vascular tone (15-18). Early in the development of chronic congestive heart failure, plasma concentrations of atrial natriuretic peptide (ANP)1 and brain natriuretic peptide increase markedly (16). These peptides have natriuretic, diuretic, and vasorelaxant effects and may decrease cardiac filling pressures and arterial pressure, increase sodium excretion, and inhibit activation of the renin-angiotensin-aldosterone system. Although these observations suggest a compensatory role for ANP in heart failure, the precise contribution of these peptides to the pathophysiology of congestive heart failure is not completely understood. We and others have shown that ANP and CNP inhibit vascular smooth muscle growth (19-21) and that ANP induces apoptosis in cultured vascular smooth muscle cells (22). We accordingly examined the effects of natriuretic peptides on cardiac myocyte apoptosis.


EXPERIMENTAL PROCEDURES

Materials

All natriuretic peptides and analogs were purchased from Peninsula Laboratories Inc. (Belmont, CA) and were >99% pure as determined by high performance liquid chromatography. RNAzol B RNA isolation kit was purchased from Tel-Test Inc. (Friendswood, TX). RT-PCR kits, and cell culture reagents were purchased from Life Technologies, Inc. Zaprinast was obtained from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA). Hoechst 33342 (H33342) and propidium iodide were purchased from Molecular Probes (Eugene, OR). DIG oligonucleotide 3'-end labeling kit and DIG luminescent detection kit were purchased from Boehringer Mannheim. All other reagents were of the highest purity available and were obtained from Sigma unless noted.

Cell Culture

Enriched cultures of myocyte and non-myocyte cells were obtained from the neonatal rat by stepwise trypsin dissociation as described previously (30). After overnight plating in minimum Eagle's medium + 5% fetal calf serum, cells were maintained in serum-free minimum Eagle's medium supplemented with transferrin and insulin as described previously (30) The final myocyte cultures contained >90% ventricular myocytes at a density of 4 × 106 cells/60-mm dish. Experiments were performed 3 days after plating. To decrease the potential effect of endogenously produced natriuretic peptides, the culture medium was replaced daily.

Non-myocytes from the same cultures were obtained by selective preplating (30). These cells (approximately 95% fibroblasts, with small percentages of smooth muscle and endothelial cells) were used at passages 1-3. For experiments, non-myocytes were plated onto 35-mm dishes or two-welled chamber slides at a density of 1 × 105 cells/cm2 in 5% serum. Following overnight attachment, the non-myocyte cultures were placed in serum-free medium as above, and experiments were performed 3 days later.

Quantitative Analysis of Apoptotic Nuclei

Cells were analyzed for apoptosis following visualization of nuclear morphology with the fluorescent DNA-binding dyes H33342 and propidium iodide. On day 3 of serum-free culture, cells were treated for various times with the indicated test compounds. The monolayers were rinsed with phosphate-buffered saline and then incubated with 5 µg/ml H33342 and 5 µg/ml propidium iodide for 30 min. Individual nuclei were visualized at × 400 using fluorescent microscopy and analyzed for apoptotic characteristics; propidium iodide was used to identify nonviable cells. Propidium iodide-positive cells with near-normal chromatin were counted as necrotic. An average of 800-1000 nuclei from random fields were analyzed for each data point. The apoptotic index (percentage of apoptotic nuclei) was calculated as (apoptotic nuclei/total nuclei) x 100%. Sample identities were concealed during scoring, and at least three samples were scored per group.

Analysis of DNA Fragmentation

Cells were treated with test compounds for 24 h on days 3-4 of culture. Nuclear DNA was isolated from treated and untreated cells following cell lysis in 300 mM NaCl, 50 mM Tris, pH 8, 25 mM EDTA containing 0.15 mg/ml proteinase K and 100 µg/ml RNase A. The lysate was incubated for 3 h at 37 °C and was then deproteinated by two phenol/chloroform/isoamyl alcohol extractions. 1 µg of DNA was used for end labeling with DIG-ddUTP and terminal transferase for 30 min at 37 °C. DNA labeling efficiency was checked by dot blot followed by detection with an alkaline phosphatase-coupled anti-digoxigenin antibody and the chemiluminescent substrate disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate. For the analysis of DNA fragmentation, digoxigenin-labeled DNA was size-fractionated in a 2% agarose gel and cleaved with brief depurination in 0.2 N HCl for 10 min and partial hydrolysis in 0.5 N NaOH, 1.5 M NaCl for 30 min followed by neutralization with 1 M Tris-HCl, 1.5 M NaCl. Following transfer to nylon membrane overnight in 20 × SSC, the DNA was visualized immunochemically as above.

Immunofluorescence Cell Staining

Desmin was used to distinguish cardiac myocytes from non-myocytes as described previously (24). Myocyte and non-myocyte cultures were plated on 2-well chamber slides and cultured for 3 days prior to 24-h treatment with ANP as described above. Cells were fixed and permeabilized with ice-cold methanol, blocked with 5% heat-inactivated horse serum/phosphate-buffered saline and sequentially incubated with anti-desmin monoclonal antibody (1:200 dilution in Tris-buffered saline, Sigma), horse anti-mouse IgG antibody conjugated to biotin (3 µg/ml, Vector Laboratories), and FITC-avidin (5 µg/ml) and H33342. The cells were visualized using fluorescence microscopy and documented by dual exposure photography.

RNA Extraction and PCR Analysis

Total cellular RNA (1 µg) was extracted using RNAzol B and analyzed by RT-PCR. Oligonucleotide primers specific for Mcl-1 were derived from the human sequence (25): forward, 5' CTG TAC AAG GGA AGC TTT 3'; reverse, 5' GAA AAG CTG TAA GAA ACA GG 3' (product: 334 base pairs). As an internal control for the semiquantitative analysis of Mcl-1, beta -actin was also amplified. beta -Actin primers were derived from the rat sequence, as described previously (23): forward, 5' TGG AGA AGA GCT ATG AGC TGC CTG 3'; reverse, 5' GTG CCA CCA GAC AGC ACT GTG TTG 3' (product: 210 base pairs). For the analysis of Mcl-1 mRNA, the RNA was reverse-transcribed and then aliquoted into two PCR tubes, one for amplification of beta -actin transcripts and the second for amplification of Mcl-1 transcripts. PCR was performed for 35 (Mcl-1) or 15 (beta -actin) cycles each at 94 °C × 1 min, 55 °C × 2 min, 72 °C × 3 min, and 1 cycle of 72 °C × 15 min. After reaction, the PCR products for Mcl-1 and beta -actin were pooled, size-separated through 1.5% agarose gel and visualized under UV light.

Statistical Analysis

Data are presented as mean ± S.E. Analysis was performed by analysis of variance followed by Fischer PLSD, p < 0.05% was considered significant.


RESULTS

Morphological Study of ANP-induced Apoptosis

To test the ability of ANP to modulate apoptosis in cardiac myocytes, enriched cultures of neonatal myocytes were treated with 1 µM ANP for 24 h on day 4 of culture and examined in situ following staining with H33342 and propidium iodide (Fig. 1A). ANP treatment caused a marked increase in the percentage of apoptotic cells (19.1% ± 1.45 versus 4.78 ± 0.65 in control cultures, n = 10, p < 0.05). The percentage of necrotic cells was identical in vehicle and ANP-treated cultures (3.25 ± 0.24% and 3.38 ± 0.52%, respectively, n = 10).


Fig. 1. Cardiac myocyte apoptosis. A, morphological characteristics. Rat cardiac myocytes were cultured on two-well Nunc chamber slides as described under "Experimental Procedures" and treated with 1 µM ANP for 24 h, then stained with the fluorescent DNA-binding dyes H33342 (5 µg/ml) and propidium iodide (5 µg/ml) and viewed under fluorescent microscopy. Open arrows indicate normal myocyte nuclei; filled arrows indicate apoptotic nuclei; and the arrowhead indicates a normal non-myocyte nuclei. B, dose dependence and peptide specificity of ANP-induced apoptosis. Cultured cardiac myocytes (squares, circles) or non-myocytes (triangles) were treated for 24 h with the indicated concentrations of vehicle, rat ANP, eel ANP (from A. japonicum), and/or the ANP analog des-(Cys-Cys) rANP. Nuclei were visualized with H33342 and propidium iodide. Apoptotic nuclei (showing typical features of condensation and fragmentation) were counted and expressed as percentage of total nuclei. The results are the means ± S.E. for an average of 800-1000 nuclei from random fields in blinded experiments. A minimum of triplicate samples were scored. * represents p < 0.05. C, ANP induces laddering of myocyte chromosomal DNA. DNA isolated from the ANP-treated (1 µM) myocytes was examined for internucleosomal cleavage, a hallmark of apoptosis. Nuclear DNA was isolated from enriched cultures of myocytes (lanes 1 and 2) and enriched cultures of non-myocytes (lanes 3 and 4) following 24 h treatment with ANP (lanes 2 and 4) or vehicle (lanes 1 and 3). The DNA was end-labeled with DIG-ddUTP and terminal transferase for 30 min prior to size fractionation on an agarose gel. Following transfer to a nylon membrane, the DNA was detected immunologically using an anti-digoxigenin antibody.
[View Larger Version of this Image (34K GIF file)]

Induction of apoptosis by ANP was dose-dependent and receptor-mediated. A maximum 4.1-fold induction was obtained at an ANP concentration of 1 µM and an approximate ED50 of 5-10 nM (Fig. 1B). This effect was not seen with two different synthetic analogs of ANP, des-(Cys-Cys) rANP and C-ANF, that bind only to the ANP clearance receptor but not the guanylyl cyclase-linked A- and B-type receptors (Fig. 1B and data not shown). Similarly, ANP isolated from the Japanese eel (Anguilla japonica), which has very low affinity for the rat ANP receptor, and exerts 20-fold less vasodepressor and natriuretic activity in the rat (26), had a correspondingly reduced effect on the induction of apoptosis in these rat myocytes (Fig. 1B). The pro-apoptotic activity of ANP is thus likely to be mediated via the A- and B-type natriuretic peptide receptors.

DNA cleavage into nucleosomal sized fragments is a hallmark of apoptotic cell death (1). DNA isolated from control myocytes exhibited a low level of DNA fragmentation, consistent with a low, basal rate of apoptosis under serum-free conditions. On the other hand, DNA from the ANP-treated myocytes was extensively fragmented, yielding the characteristic nucleosomal "ladder" (Fig. 1C).

ANP-induced Apoptosis Is Myocyte-specific

The non-myocytes from the neonatal heart do not undergo ANP-induced apoptosis. Enriched cultures of primary myocyte contain a small percentage (<= 5%) of non-myocytes, of which >90% are fibroblasts and ~5% are smooth muscle cells by morphological and immunocytochemical criteria (24).2 In confirmation of an earlier report (27), RT-PCR analysis showed that both myocyte and non-myocyte populations express all three known isoforms of the natriuretic receptors at similar levels (data not shown). Thus, both cell populations may be competent to respond to ANP. However, ANP did not appear to induce apoptosis in the non-myocyte cultures. Under basal conditions, the apoptotic index of the non-myocyte cultures was very low (<1%) and was unchanged by ANP as assessed by nuclear morphology (Fig. 1B) or DNA laddering assay (Fig. 1C). Thus, although non-myocytes express receptors for natriuretic peptides, they appear to be resistant to ANP-induced apoptosis.

To confirm this observation, we directly examined the apoptotic cells in non-myocyte and myocyte cultures with a monoclonal mouse anti-desmin antibody and the nuclear stain H33342 (Fig. 2). Desmin expression is generally a muscle-restricted protein, and only the cardiac myocytes in these mixed primary cultures are desmin-positive (24). Greater than 95% of the cells in these cultures were desmin-positive and possessed other morphological features of cardiac myocytes (Fig. 2, B and C). In contrast, fewer than 1% of the cells in the non-myocyte cultures were desmin-positive, and these typically represented contaminating myocytes. Virtually all apoptotic cells under basal conditions and after ANP treatment were also desmin-positive. We conclude that, in these mixed cultures, ANP-induced apoptosis is limited to cardiac myocytes.


Fig. 2. Immunohistochemical characterization of apoptotic cells. Primary cultures of cardiac myocytes and selectively plated non-myocytes were treated with 1 µM ANP for 24 h. The cells were then fixed with ice-cold methanol and incubated with anti-desmin antibody and a FITC-tagged secondary antibody as described under "Experimental Procedures." The cells were then incubated with H33342 and visualized by fluorescence microscopy using UV and FITC spectrum filters. A-C, myocytes treated with ANP. D-F, non-myocytes treated with ANP. For each cell type, the three panels show the same field of cells stained with Hoechst 33342 (A and D), FITC-anti-desmin (B and E) and both Hoechst and anti-desmin (C and F). In A-C, open arrows indicate normal myocyte nuclei; filled arrows indicate apoptotic nuclei; and the arrowhead indicates a normal non-myocyte nuclei.
[View Larger Version of this Image (74K GIF file)]

Non-myocytes Do Not Influence ANP-induced Myocyte Apoptosis

It is possible that ANP-induced apoptosis is an indirect effect mediated by non-myocytes (or myocytes) through paracrine/autocrine mechanisms. To rule out this possibility, we conducted a series of experiments in which media were conditioned by incubation with myocytes, non-myocytes, or in empty tissue culture dishes, for varying intervals after the addition of ANP. The results are summarized in Fig. 3, A and B. Neither myocyte nor non-myocyte-derived media altered basal rates of myocyte apoptosis after 12 h of conditioning, regardless of ANP treatment (Fig. 3A). Furthermore, the ability of ANP-treated non-myocyte conditioned media to induce apoptosis at earlier time points exactly paralleled that of ANP-containing medium maintained in an empty tissue culture dish. Induction of apoptosis by either medium was maximal immediately after ANP addition and declined in a linear fashion over a further 8 h of conditioning (Fig. 3B). These data suggest that all of the apoptotic potential of the conditioned media can be attributed to the added ANP peptide. Thus, it is unlikely that non-myocytes contribute to the effects of ANP on cardiac myocyte apoptosis.


Fig. 3. Conditioned medium from non-myocytes do not induce myocyte apoptosis. A, medium with or without ANP (1 µM) was incubated with myocytes, non-myocytes, or empty plastic Petri dishes for 12 h. The conditioned medium (CM) was removed and added to myocytes for an additional 12 h. In addition, one set of myocytes were treated with media containing fresh ANP (1 µM). The percent apoptotic cells was then measured with H33342. Note that only the myocytes treated with fresh ANP exhibited an increase in the incidence of apoptosis. The results are the means ± S.E. for an average of 800-1000 nuclei from random fields in blinded experiments. A minimum of triplicate samples were scored. B, as in A, medium with or without ANP (1 µM) was added to non-myocyte cultures or to empty Petri dishes. At various times, the medium was collected and placed onto myocytes, and the myocytes were incubated for an additional 12 h. For comparison, separate groups of myocytes were incubated with vehicle or fresh ANP (1 µM), also for 12 h. The percent of apoptotic cells was measured with H33342. Note that the ability of conditioned media from ANP-treated non-myocytes to increase the incidence of apoptosis was indistinguishable from the ability of ANP incubated alone at 37 °C. The results are the means ± S.E. for an average of 800-1000 nuclei from random fields in blinded experiments. A minimum of triplicate samples were scored.
[View Larger Version of this Image (35K GIF file)]

cGMP Can Induce Myocyte Apoptosis

As shown above, the ANPC receptor is not likely to be a mediator of ANP-induced apoptosis. Of the two other ANP receptor subtypes, ANPA and ANPB, only the former binds ANP with high affinity (28). The dose-response relationship presented in Fig. 1 is thus consistent with an action mediated by the ANPA receptor. The ANPA receptor is coupled to generation of cGMP, suggesting that guanylyl cyclase and cGMP may participate in ANP-induced apoptosis. To confirm this, we tested a series of cGMP modulating agents. Sodium nitroprusside (10-6 to 10-3 M), a guanylate cyclase-activating nitric oxide donor, also induced cardiac myocyte apoptosis (Fig. 4A). Zaprinast, a cGMP-specific phosphodiesterase inhibitor that has been shown to potentiate other effects of ANP on cardiac myocytes (29), strongly potentiated the induction of apoptosis by a submaximal dose of ANP (1 nM, Fig. 4B; 3.3-fold versus 2.1-fold in the absence of zaprinast.) Finally, the cell-permeable cGMP analog 8-bromo-cGMP caused a dose-dependent induction of apoptosis at 24 h, with a maximum of 3.4-fold over control at 100 µM and an approximate EC50 of 0.08 µM (Fig. 4A). Taken together, these results strongly suggest that ANP-induced apoptosis is mediated by a guanylyl cyclase linked receptor via elevation of intracellular cGMP.


Fig. 4. ANP-induced apoptosis is mediated by cGMP. A, induction of apoptosis by the cGMP agonist sodium nitroprusside and by cell permeable analogs of cGMP. Cells were treated for 24 h with the indicated concentrations of 8-bromo-cGMP (1 nM to 1 µM) or of sodium nitroprusside, which increases cGMP via generation of NO, independent of natriuretic peptide receptors. * represents p < 0.05. B, potentiation of ANP-induced apoptosis by zaprinast (Zap). Cells were treated with a submaximal concentration of ANP (1 nM) with and without the cGMP-selective phosphodiesterase inhibitor zaprinast (10 nM). * represents p < 0.05.
[View Larger Version of this Image (17K GIF file)]

ANP-induced Apoptosis Is Blocked by Norepinephrine

Norepinephrine is an adrenergic agonist known to stimulate hypertrophic growth of cultured myocytes (30). Since growth factors frequently act in opposition to apoptosis in other systems, we asked whether norepinephrine would have an inhibitory effect on basal and ANP-induced cardiac myocyte apoptosis. Treatment of cultures with norepinephrine alone for 24 h did not influence the basal rate of apoptosis (Fig. 5A). However, norepinephrine reduced the apoptotic index to control levels in ANP-stimulated myocytes (Fig. 5A).


Fig. 5. Adrenergic stimulation inhibits ANP-induced apoptosis. A, norepinephrine (NE) blocks ANP-induced apoptosis. Cells were treated with ANP (1 µM) or vehicle for 24 h in the presence or absence of norepinephrine (1 µM). Apoptotic cells were detected and scored as indicated above. B, beta -adrenergic stimulation blocks apoptosis. Cultured myocytes were treated with ANP (1 µM) or ANP plus norepinephrine (NE) (1 µM) in the presence or absence of an alpha 1-adrenergic receptor antagonist (terazosin (T), 4 µM) or a beta -adrenergic receptor antagonist (propranolol (P), 4 µM). Twenty-four hours later, apoptotic cells were detected and scored as indicated above. C, beta -adrenergic blockade of apoptosis is mediated by cAMP-dependent protein kinase. Cultured myocytes were treated with the indicated combinations of ANP (1 µM), the beta -adrenergic receptor agonist isoproterenol (Iso) (4 µM), the calcium channel blocker nifedipine (Nif) (5 µM), or a specific protein kinase A inhibitor (KT5720 (KT), 0.5 µM). D, Bt2cAMP (db cAMP) blocks ANP-induced apoptosis. Vehicle or ANP treated cells were treated with the cell permeable analog of cAMP (Bt2cAMP) at 1 µM for 24 h. Apoptotic cells were detected and scored as indicated above.
[View Larger Version of this Image (22K GIF file)]

Norepinephrine influences myocyte growth in culture via both alpha 1 and beta -adrenoreceptors (30-34), depending on the extent of cell contact. To examine the receptor class mediating the anti-apoptotic effect of norepinephrine, myocytes were treated with ANP and norepinephrine alone or in combination with subtype-selective adrenergic receptor antagonists, either propranolol (beta -selective) or terazosin (alpha 1-selective). Only the beta -adrenoreceptor antagonist propranolol blocked the anti-apoptotic effect of norepinephrine (Fig. 5B), suggesting that this effect was mediated by a beta -adrenoreceptor. Neither antagonist had independent effects on basal or ANP-induced apoptosis (Fig. 5B).

beta -Adrenoreceptor-mediated signaling in cardiac myocytes involves both an activation of the adenylate cyclase/cAMP generating pathway and a G protein mediated increase in extracellular calcium influx (31). To further analyze the protective effects of beta -adrenergic stimulation, we measured ANP-induced apoptosis in the presence of various combinations of isoproterenol, a beta -adrenergic agonist; nifedipine, an L-type calcium entry antagonist; and KT5720, a highly selective inhibitor of cAMP-dependent protein kinase (protein kinase A, Ref. 31). As predicted, isoproterenol (4 µM) was equipotent to norepinephrine in blocking ANP-induced apoptosis. Interestingly, inhibition of protein kinase A, but not of calcium entry, blunted the anti-apoptotic effects of isoproterenol, suggesting a cAMP-dependent effect (Fig. 5C). Consistent with these results, the membrane-permeable cAMP analog dibutyryl cAMP (Bt2cAMP) also inhibited ANP-induced apoptosis (Fig. 5D). The effects of Bt2cAMP were dose-dependent and were reversed at concentrations higher than 1 mM (not shown). These results suggest that the anti-apoptotic effect of beta -adrenergic stimulation is mediated through activation of protein kinase A and does not require calcium influx.

ANP-induced Apoptosis Is Associated with a Decrease in Mcl-1 Expression

Apoptosis is modulated by the balance of pro- and anti-apoptotic proteins, including members of the Bcl-2 family (35, 36). Mcl-1, a Bcl-2 homologue (25), has been shown to inhibit apoptosis in Chinese hamster ovary cells (37) and appears to be expressed at relatively high levels in the heart in comparison with other Bcl-2-related proteins (38). PCR analysis revealed that ANP induced a time-dependent decrease in Mcl-1 mRNA levels that reached a nadir at 16 h and returned to base line at 48 h (Fig. 6). Control PCR-amplified beta -actin transcript levels did not change over the same interval. Mcl-1 transcripts were not detectable in non-myocytes using the same protocol (not shown).


Fig. 6. ANP inhibits Mcl-1 expression in cardiac myocytes. Cultured myocytes were treated with 1 µM ANP for various times from 0 to 48 h. Total RNA was isolated and analyzed for Mcl-1 and beta -actin mRNA levels by RT-PCR. Products from the reverse transcription were split into two aliquots and beta -actin, and Mcl-1 mRNA transcripts were amplified separately by PCR (for 15 and 35 cycles, respectively). PCR products from the two reactions were mixed and size separated together on 2% agarose. The last lane shows the results of similar RT-PCR amplification of total RNA from neonatal heart tissue.
[View Larger Version of this Image (51K GIF file)]


DISCUSSION

In this paper, we report a novel direct effect of ANP on cardiac myocytes, the induction of apoptosis. ANP-induced apoptosis is dose-dependent, receptor-specific, and involves natriuretic peptide receptor-mediated increases in cGMP. Furthermore, this apoptosis is antagonized by catecholamine-induced increases in cAMP. Given the previously documented ability of the neonatal rat cardiac myocyte cell culture system to identify direct cardiotrophic properties of both soluble and mechanical factors (39-44), our results provide a basis for further investigation of this property of ANP in animal models of cardiac growth and failure.

A number of neurohumoral and autocrine substances, including endothelin, angiotensin II, and norepinephrine, are known to stimulate cardiac myocyte and smooth muscle growth (20-22, 41-45). That there might exist a complementary ANP-mediated mechanism to inhibit growth in these cells is perhaps not surprising. Growth inhibition is often accompanied by an increased probability of apoptosis, while growth-promoting agents tend also to promote survival. For example, angiotensin II inhibits smooth muscle cell apoptosis (46), while nitric oxide, transforming growth factor beta 1 and ANP, all potent inhibitors of smooth muscle cell growth, induce apoptosis in these cells (46, 47, 22). Significantly, nitric oxide is thought to induce apoptosis in cardiac myocytes via cGMP generation, similar to ANP (6). Since the heart is the major source of this natriuretic peptide, we postulate that ANP may be an autocrine growth inhibitor, opposing the effect of autocrine and paracrine growth factors.

Enhanced production and secretion of ANP accompanies cardiac myocyte hypertrophy. Although ANP has been shown to inhibit growth of cardiac fibroblasts in culture (48), as yet there is only indirect evidence that natriuretic peptides influence myocyte growth. Transgenic mice overexpressing ANP exhibit a 27% reduction in total heart weight (49) and appear to have a blunted right ventricular hypertrophic response to hypoxia-induced pulmonary hypertension (50), while mice homozygous for disruption of the pro-ANP gene exhibit a significant increase in the ratio of heart weight to body weight (51). Stabilization of ANP with alatriopril, a mixed angiotensin converting enzyme/atriopeptidase inhibitor, resulted in a greater reduction of compensatory hypertrophy compared with an angiotensin converting enzyme inhibitor alone in a rat model of myocardial infarction (52). Although some of these observations may be due in part to ANP effects on blood pressure, it is clear that the net effect of ANP on myocardium in vivo is one that limits myocyte growth.

Another interesting feature of our results is the inverse relationship between cAMP and cGMP with respect to apoptosis. cGMP itself may either inhibit (53-56), promote (6, 22, 46, 57-61), or have no effect (see Figs. 1 and 2) on apoptosis, depending on the cell type. In this case, ANP induction of apoptosis via cGMP clearly involves cardiac myocyte-specific signal transduction mechanisms. The beta -adrenergic receptor-mediated trophic effects of norepinephrine are likewise restricted to cardiac myocytes in these cultures (31). beta -Adrenergic stimulation antagonized the pro-apoptotic effects of ANP through a cAMP/protein kinase A-dependent mechanism associated with morphological evidence of cell growth (not shown). cAMP and cGMP exhibit a reciprocal relationship in studies of beta -adrenoreceptor-stimulated cardiac contractility (62), and the molecular mediators of these effects on contractility have been well characterized. However, the molecular targets for cyclic nucleotide-modulated cell fate decisions are unknown.

Our data suggests that the anti-apoptotic Bcl-2 homologue, Mcl-1 (25, 35-38), may be one such target in the cardiac myocyte. The Bcl-2 homologue Mcl-1 was initially identified as a protein up-regulated during the differentiation of a monocytoid cell line, ML-1 cells (25), and has been shown to inhibit apoptosis in Chinese hamster ovary cells (37). Interestingly, Mcl-1 is expressed at high levels in the heart (38), an observation confirmed in this study. Although these studies do not establish a causal relationship between the ANP-induced fall in Mcl-1 transcripts and increased apoptosis, it is consistent with the hypothesis that Mcl-1 expression is one determinant of the probability of apoptosis in the cardiac myocyte, along with many other factors (25, 35, 36). Further studies on the role of Mcl-1 and related proteins in ANP-induced apoptosis are in progress.

A number of neuroendocrine effectors, including ANP, brain natriuretic peptide, renin, angiotensin II, norepinephrine, and arginine vasopressin, are present at high levels early in the development of heart failure and may play an important role in its progression (15-18). Our data suggest that the sustained elevation of ANP may have direct effects on the cardiac myocyte independent of its hemodynamic actions. Whether the cumulative effects of ANP and other agents are harmful or beneficial in vivo is likely to depend on a balance of external and intracellular factors. Further studies of this effect of ANP will help to illuminate the precise molecular effectors and targets of growth restriction and apoptosis in the cardiac myocyte.


FOOTNOTES

*   This work was presented in part at the American College of Cardiology National Meeting, Orlando, FL, March 24-27, 1996, and the American Heart Association Scientific Sessions, New Orleans, LA, November 10-13, 1996. This work was supported by National Institutes of Health Grants HL42663 (to R. E. P.) and HL49891 (to N. H. B.) and Training Grant HL07708 for Vascular Medicine and Biology.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.
§   Current address: Cardiovascular Research Dept., Genentech, Inc., 460 Point San Bruno Blvd., South San Francisco, CA 94080.
par    Current address: Dept. of Molecular and Cellular Pharmacology, University of Miami School of Medicine (R-189), P. O. Box 016189, Miami, FL 33101. E-mail: nhb{at}chroma.med.miami.edu.
**   To whom correspondence should be addressed. Current address: Laboratory of Genetic Physiology, Cardiovascular Research, Thorn-12, Brigham and Women's Hospital, 75 Francis St., Boston, MA 00000. Tel.: 617-732-8799; Fax: 617-975-0995; E-mail: rpratt{at}bustoff.bwh.harvard.edu.
1   The abbreviations used are: ANP, atrial natriuretic peptide; RT-PCR, reverse transcription-polymerase chain reaction; DIG, digoxigenin; cGMP, cyclic GMP; FITC, fluorescein isothiocyanate; Bt2cAMP, dibutyryl cyclic AMP.
2   N. H. Bishopric, unpublished data.

ACKNOWLEDGEMENTS

We thank Barbara Sato for technical assistance and Keith Webster, Pedro Trindade, and Howard Hutchinson for discussion and advice.


REFERENCES

  1. Wyllie, A. H., Kerr, J. F. R., and Currie, A. R. (1980) Int. Rev. Cytol. 68, 251-306 [Medline] [Order article via Infotrieve]
  2. Bing, O. H. L. (1994) J. Mol. Cell. Cardiol. 26, 943-948 [CrossRef][Medline] [Order article via Infotrieve]
  3. James, T. N., St. Martin, E., Willis, P. W., III, and Lohr, T. O. (1996) Circulation 93, 1424-1438 [Abstract/Free Full Text]
  4. Cheng, W., Kajstura, J., Nitahara, J. A., Li, B., Reiss, K., Liu, Y., Clark, W. A., Krajewski, S., Reed, J. C., Olivetti, G., and Anversa, P. (1996) Exp. Cell Res. 226, 316-327 [CrossRef][Medline] [Order article via Infotrieve]
  5. Cheng, W., Li, B., Kajstura, J., Li, P., Wolin, M. S., Sonnenblick, E. H., Hintze, T. H., Olivetti, G., and Anversa, P. (1995) J. Clin. Invest. 96, 2247-2259 [Medline] [Order article via Infotrieve]
  6. Pinsky, D. J., Cai, B., Yang, X., Rodriguez, C., Sciacca, R. R., and Cannon, P. J. (1995) J. Clin. Invest. 95, 677-685 [Medline] [Order article via Infotrieve]
  7. Teiger, E., Than, V. D., Richard, L., Wisnewsky, C., Tea, B. S., Gaboury, L., Tremblay, J., Schwartz, K., and Hamet, P. (1996) J. Clin. Invest. 97, 2891-2897 [Abstract/Free Full Text]
  8. Mallat, Z., Tedgui, A., Fontaliran, F., Frank, R., Durigon, M., and Fontaine, G. (1996) N. Engl. J. Med. 335, 1190-1196 [Abstract/Free Full Text]
  9. Narula, J., Haider, N., Virmani, R., DiSalvo, T. G., Kolodgie, F. D., Hajjar, R. J., Schmidt, U., Semigran, M. J., Dec, G. W., and Khaw, B. (1996) N. Engl. J. Med. 335, 1182-1189 [Abstract/Free Full Text]
  10. Kirshenbaum, L. A., and Schneider, M. D. (1995) J. Biol. Chem. 270, 7791-7794 [Abstract/Free Full Text]
  11. James, T. N. (1994) Circulation 90, 556-573 [Abstract]
  12. Gottleib, R. A., Burleson, K. O., Kloner, R. A., Babior, B. M., and Engler, R. L. (1994) J. Clin. Invest. 94, 1621-1628 [Medline] [Order article via Infotrieve]
  13. Tanaka, M., Ito, H., Adachi, S., Akimoto, H., Nishikawa, T., Kasajima, T., Marumo, F., and Hiroe, M. (1994) Circ. Res. 75, 426-433 [Abstract]
  14. Kajstura, J., Cheng, W., Reiss, K., Clark, W. A., Sonnenblick, E. H., Krajewski, S., Reed, J. C., Olivetti, G., and Anversa, P. (1996) Lab. Invest. 74, 86-107 [Medline] [Order article via Infotrieve]
  15. Morgan, H. E. (1993) Circulation 87, 4-6
  16. Wei, C. M., Heublein, D. M., Perrella, M. A., Lerman, A., Rodeheffer, R. J., McGregor, C. G., Edwards, W. D., Schaff, H. V., and Burnett, J. C., Jr. (1993) Circulation 88, 1004-1009 [Abstract]
  17. Francis, G. S., and Chu, C. (1995) Curr. Opin. Cardiol. 10, 260-267 [Medline] [Order article via Infotrieve]
  18. Coats, A. J., and Adamopoulos, S. (1994) Cardiovasc. Drugs Ther. 8, 685-692 [Medline] [Order article via Infotrieve]
  19. Itoh, H., Pratt, R., and Dzau, V. J. (1990) J. Clin. Invest. 86, 1690-1697 [Medline] [Order article via Infotrieve]
  20. Cahill, P. A., and Hassid, A. (1993) J. Cell. Physiol. 154, 28-38 [Medline] [Order article via Infotrieve]
  21. Porter, J. G., Catalano, R., McEnroe, G., Lewicki, J. A., and Protter, A. A. (1992) Am. J. Physiol. 263, C1001-C1006 [Abstract/Free Full Text]
  22. Trindade, P., Hutchinson, H. G., Pollman, M. J., Gibbons, G. H., and Pratt, R. E. (1995) Circulation 92, I-696 (abstr.)
  23. Bodí, I., Bishopric, N. H., Discher, D. J., Wu, X., and Webster, K. A. (1995) Cardiovasc. Res. 30, 975-984 [CrossRef][Medline] [Order article via Infotrieve]
  24. Antin, P. B., Mar, J. H., and Ordahl, C. P. (1988) BioTechniques 6, 640-649 [Medline] [Order article via Infotrieve]
  25. Kozopas, K. M., Yang, T., Buchan, H. L., Zhou, P., and Craig, R. W. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3516-3520 [Abstract]
  26. Takei, Y., Takahashi, A., Watanabe, T. X., Nakajima, K., and Sakakibara, S. (1989) Biochem. Biophys. Res. Commun. 164, 537-543 [Medline] [Order article via Infotrieve]
  27. Lin, X., Hanze, J., Heese, F., Sodmann, R., and Lang, R. E. (1995) Circ. Res. 77, 750-758 [Abstract/Free Full Text]
  28. Koller, K. J., Lowe, D. G., Bennett, G. L., Minamino, N., Kangawa, K., Matsuo, H., and Goeddel, D. V. (1991) Science 252, 120-123 [Medline] [Order article via Infotrieve]
  29. Clemo, H. F., and Baumgarten, C. M. (1995) Circ. Res. 77, 741-749 [Abstract/Free Full Text]
  30. Bishopric, N. H., and Kedes, L. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2132-2136 [Abstract]
  31. Bishopric, N. H., Sato, B., and Webster, K. A. (1992) J. Biol. Chem. 267, 20932-20936 [Abstract/Free Full Text]
  32. Milano, C. A., Allen, L. F., Rockman, H. A., Dolber, P. C., McMinn, T. R., Chien, K. R., Johnson, T. D., Bond, R. A., and Lefkowitz, R. J. (1994) Science 264, 582-586 [Medline] [Order article via Infotrieve]
  33. Milano, C. A., Dolber, P. C., Rockman, H. A., Bond, R. A., Venable, M. E., Allen, L. F., and Lefkowitz, R. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10109-10113 [Abstract/Free Full Text]
  34. Knowlton, K. U., Michel, M. C., Itani, M., Shubeita, H. E., Ishihara, K., Brown, J. H., and Chien, K. R. (1993) J. Biol. Chem. 268, 15374-15380 [Abstract/Free Full Text]
  35. Oltvai, Z. N., Milliman, C. L., and Korsmeyer, S. J. (1993) Cell 74, 609-619 [Medline] [Order article via Infotrieve]
  36. Kiefer, M. C., Brauer, M. J., Powers, V. C., Wu, J. J., Umansky, S. R., Tomei, L. D., and Barr, P. J. (1995) Nature 374, 736-739 [CrossRef][Medline] [Order article via Infotrieve]
  37. Reynolds, J. E., Yang, T., Qian, L., Jenkinson, J. D., Zhou, P., Eastman, A., and Craig, R. W. (1994) Cancer Res. 54, 6348-6352 [Abstract]
  38. Krajewski, S., Bodrug, S., Krajewska, M., Shabaik, A., Gascoyne, R., Berean, K., and Reed, J. C. (1995) Am. J. Pathol. 146, 1309-1319 [Abstract]
  39. Chien, K. R., Knowlton, K. U., Zhu, H., and Chien, S. (1991) FASEB J. 5, 3037-3046 [Abstract/Free Full Text]
  40. Parker, T. G. (1993) Herz. 18, 245-255 [Medline] [Order article via Infotrieve]
  41. Yamazaki, T., Komuro, I., and Yazaki, Y. (1995) J. Mol. Cell. Cardiol. 27, 133-140 [Medline] [Order article via Infotrieve]
  42. Sadoshima, J., Xu, Y., Slayter, H. S., and Izumo, S. (1993) Cell 75, 977-984 [Medline] [Order article via Infotrieve]
  43. Simpson, P. (1985) Circ. Res. 56, 884-894 [Abstract]
  44. Sadoshima, J., and Izumo, S. (1993) Circ. Res. 73, 413-423 [Abstract]
  45. Koibuchi, Y., Lee, W. E., Gibbons, G. H., and Pratt, R. E. (1993) Hypertension 21, 1046-1050 [Abstract]
  46. Pollman, M. J., Yamada, T., Moriuchi, M., and Gibbons, G. H. (1996) Circ. Res. 79, 748-756 [Abstract/Free Full Text]
  47. Pollman, M. J., and Gibbons, G. H. (1995) FASEB J. 9, A871 (Abstr. 5056)
  48. Cao, L., and Gardner, D. G. (1995) Hypertension 25, 227-234 [Abstract/Free Full Text]
  49. Klinger, J. R., Petit, R. D., Curtin, L. A., Warburton, R. R., Wrenn, D. S., Steinhelper, M. E., Field, L. J., and Hill, N. S. (1993) J. Appl. Physiol. 75, 198-205 [Abstract]
  50. Barbee, R. W., Perry, B. D., Re, R. N., Murgo, J. P., and Field, L. J. (1994) Circ. Res. 74, 747-751 [Abstract]
  51. John, S. W. M., Krege, J. H., Oliver, P. M., Hagaman, J. R., Hodgin, J. B., Pang, S. C., Flynn, T. G., and Smithies, O. (1995) Science 267, 679-681 [Medline] [Order article via Infotrieve]
  52. Bralet, J., Marie, C., Mossiat, C., Lecomte, J. M., Gros, C., and Schwartz, J. C. (1994) J. Pharmacol. Exp. Ther. 270, 8-14 [Abstract]
  53. Beauvais, F., Michel, L., and Dubertret, L. (1995) FEBS Lett. 361, 229-232 [CrossRef][Medline] [Order article via Infotrieve]
  54. Genaro, A. M., Hortelano, S., Alvarez, A., Martinez, C., and Bosca, L. (1995) J. Clin. Invest. 95, 1884-1890 [Medline] [Order article via Infotrieve]
  55. Sandvig, K., and van Deurs, B. (1992) Exp. Cell Res. 200, 253-262 [Medline] [Order article via Infotrieve]
  56. Portera-Cailliau, C., Sung, C., Nathans, J., and Adler, R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 974-978 [Abstract]
  57. Blanco, F., Ochs, R., Schwarz, H., and Lotz, M. (1995) Am. J. Pathol. 146, 75-85 [Abstract]
  58. Terenzi, F., Diaz-Guerra, M. J. M., Casado, M., Hortelano, S., Leoni, S., and Bosca, L. (1995) J. Biol. Chem. 270, 6017-6021 [Abstract/Free Full Text]
  59. Xie, K., Huang, S., Dong, Z., Juang, S. H., Gutman, M., Xie, Q. W., Nathan, C., and Fidler, I. J. (1995) J Exper. Med 181, 1333-43 [Abstract]
  60. Kitajima, I., Kawahara, K., Nakajima, T., Soejima, Y., Matsuyama, T., and Maruyama, I. (1994) Biochem. Biophys. Res. Commun. 204, 244-251 [CrossRef][Medline] [Order article via Infotrieve]
  61. Ankarcrona, M., Dypbukt, J., Brune, B., and Nicotera, P. (1994) Exp. Cell Res. 213, 172-177 [CrossRef][Medline] [Order article via Infotrieve]
  62. Hare, J. M., and Colucci, W. S. (1995) Prog. Cardiovasc. Dis. 38, 155-166 [Medline] [Order article via Infotrieve]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.