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(Circulation. 2000;102:344.)
© 2000 American Heart Association, Inc.

Basic Science Reports

ß-Adrenergic Receptor Subtypes Differentially Affect Apoptosis in Adult Rat Ventricular Myocytes

Michael Zaugg, MD; Weimin Xu, MD; Eliana Lucchinetti, MS; Saiyid A. Shafiq, PhD; Nasir Z. Jamali, MD; M. A. Q. Siddiqui, PhD

From the Department of Anesthesiology (M.Z.), Department of Neurology (W.X., S.A.S.), and Department of Anatomy and Cell Biology and Center for Cardiovascular and Muscle Research (N.Z.J., M.A.Q.S.), Health Science Center at Brooklyn, State University of New York, and the Laboratory for Soft Tissue Research, Hospital for Special Surgery (E.L.), New York, NY.

Correspondence to Dr M.A.Q. Siddiqui, Professor and Chairman, Department of Anatomy and Cell Biology, State University of New York, 450 Clarkson Ave, Box 5, Brooklyn, NY 11203. E-mail msiddiqui{at}netmail.hscbklyn.edu

	Abstract

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Background—Catecholamine-induced apoptosis is mediated by activation of the ß-adrenergic signaling pathway. We tested the hypothesis that ß₁- and ß₂-adrenergic receptor (AR) subtypes differentially affect apoptosis in adult rat ventricular myocytes in vitro.

Methods and Results—Myocytes were first exposed to norepinephrine (NE) alone (10 µmol/L) or NE+atenolol (AT) (10 µmol/L) for 12 hours. AT, a ß₁-selective AR antagonist, abolished the NE-induced increase in nick end-labeling (TUNEL)–positive cells compared with control (NE, 33±3% versus control, 3±1%, P<0.0001; NE+AT, 4±2% versus control, 3±1%, P=0.98). Annexin V staining, DNA laddering, and caspase activity determinations corroborated these results. Subsequent experiments under prazosin treatment established the apoptosis dose-response curves for the increasingly ß₂-selective AR agonists isoproterenol (ISO) (ß₁ß₂) and albuterol (ALB) (ß₂>ß₁). ISO and ALB induced significantly less apoptosis than NE (ß₁>ß₂) at equimolar concentrations as assessed by TUNEL staining [1 µmol/L: NE (8±2%)ISO (7±1%)>ALB (2±1%); 10 µmol/L: NE (35±2%)>ISO (23±1%)>ALB (3±1%); 100 µmol/L: NE (50±2%)>ISO (29±2%)>ALB (14±1%), P<0.0001 except for NE versus ISO at 1 µmol/L with P=0.62]. ALB-induced apoptosis at 100 µmol/L was abolished by AT (10 µmol/L), indicating a ß₁AR-mediated effect. Importantly, ICI 118551 (0.1 µmol/L), a highly selective ß₂AR antagonist, did not decrease the percentage of NE-, ISO-, and ALB-induced apoptosis. Reverse transcription–polymerase chain reaction studies revealed that AT completely reversed the ß-adrenergic signaling–induced changes in the Bcl-2–to-Bax ratio.

Conclusions—These observations provide evidence that ßAR-mediated apoptotic death signaling is largely dissociated from ß₂ARs and selectively mediated by ß₁ARs in adult rat ventricular myocytes.

Key Words: receptors, adrenergic, beta • catecholamines • apoptosis • heart failure

	Introduction

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Recent studies provide evidence that ß₁- and ß₂-adrenergic receptor (AR) subtypes coexist in cardiomyocytes^{1 2} but modulate cardiac function differently and exert physiological responses by distinct signal transduction pathways. In particular, ß₂AR-mediated inotropy and increase in intracellular Ca²⁺ are dissociated from bulk cytosolic cAMP production and occur without increasing phosphorylation of critical cytoplasmic proteins.^{3 4 5} Furthermore, differences between ß₁- and ß₂-adrenergic stimulation were noted in the manifestation of diastolic Ca²⁺ loading and diastolic Ca²⁺ oscillations, cell resting length, and G protein coupling characteristics.^{6 7 8}

Enhanced activation of the ß-adrenergic signaling pathway has been shown to cause necrotic^{9 10} as well as apoptotic^{11 12} death of cardiac myocytes. Accordingly, chronic heightened adrenergic drive is considered to play a pivotal role in the ongoing apoptotic cardiomyocyte cell death that leads to architectural rearrangement of myocytes, ventricular dilatation, and decreased force development.^{13 14 15} Circulating catecholamine levels are reliable predictors of the severity and outcome of congestive heart failure and support the neurohormonal hypothesis of the progression of congestive heart failure.¹⁶

Induction of apoptosis through the ß₁AR subtype and its inhibition by ß₂ARs in cardiomyocytes was reported recently.¹⁷ This finding is of great clinical interest, because selective pharmacological activation of ß₂AR-mediated inotropy or its overexpression through gene therapy might be used as a novel therapeutic approach in the failing heart.^{18 19} The present study was also undertaken to determine the significance of ß₁AR and ß₂AR stimulation on apoptotic cell death in adult rat ventricular myocytes (ARVMs). The experimental results of the present study provide further evidence that ß-adrenergic apoptotic cell death signaling is largely dissociated from ß₂ARs and selectively mediated by ß₁ARs in ARVMs in vitro.

	Methods

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Preparation of Isolated Cardiac Myocytes
Ca²⁺-tolerant ARVMs were isolated from hearts of male Sprague-Dawley rats (300 to 350 g) by standard enzymatic techniques.²⁰ The isolated ARVMs were resuspended in serum-free defined culture medium consisting of DMEM with 2 mg/mL BSA, 2 mmol/L L-carnitine, 5 mmol/L creatine, and 5 mmol/L taurine supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B. ARVMs, at a density of 100 to 150 cells/mm², were grown in 60-mm plastic culture dishes (Falcon) or on 22x22-mm glass coverslips (Fisher) precoated with laminin (1 µg/cm², Sigma) placed in 35-mm plastic culture dishes. The cells were then treated according to the specific treatment regimens over 12 hours as indicated below. Protocols were approved by the Institutional Animal Care and Use Committee.

Cell Treatments
All dishes were supplemented with fresh ascorbic acid (0.1 mmol/L, Sigma) immediately before treatment. Norepinephrine (l-NE, 10 µmol/L, Sigma) alone or in combination with atenolol (AT) (10 µmol/L, Sigma, added 30 minutes before the addition of l-NE) was added to culture dishes for 12 hours. In separate experiments ARVMs were exposed to increasing concentrations of l-NE, isoproterenol (ISO), or albuterol (ALB) (1, 10, and 100 µmol/L, Sigma) 30 minutes after addition of prazosin (0.1 µmol/L, Sigma). In some experiments, dl-propranolol (PRO, 2 µmol/L, Sigma), AT (10 µmol/L), and ICI 118551 (0.1 µmol/L) were added to the dishes along with prazosin (0.1 µmol/L) 30 minutes before the addition of l-NE (10 µmol/L), ISO (10 µmol/L), or ALB (100 µmol/L). The treatment protocols and the concentrations of the reagents used were established previously.^{11 21 22 23}

DNA Isolation and Electrophoresis
Internucleosomal DNA fragmentation was assessed by use of an isotopic TACS Apoptotic DNA laddering kit (R&D Systems). DNA samples (1 µg) from control, NE-treated, and NE+AT-treated cultures were labeled by enzymatic assay with terminal deoxynucleotidyl transferase in a buffer containing 0.5 µCi [³²P]dCTP (New England Nuclear). After incubation for 10 minutes at room temperature, the samples were analyzed on a 1.5% agarose gel. A labeled 1-kb DNA ladder was used as marker.

Annexin V/Propidium Iodide Staining
The viability of control, NE-treated, and NE+AT-treated myocytes was assessed by staining with the phosphatidylserine-binding FITC-labeled annexin V and the nucleic acid–binding propidium iodide (Fluos Staining kit, Boehringer Mannheim). Whereas FITC-labeled annexin V detects phosphatidylserine translocation on the cell surface, a hallmark of early apoptosis, late apoptosis and necrosis show additional positive nuclear staining with propidium iodide.²⁴ The percentages of early apoptotic cells with only green sarcolemmal staining (annexin V–FITC excited at 450 to 480 nm) and of late apoptotic and necrotic cells with both green sarcolemmal staining and red nuclear staining (propidium iodide excited at 510 to 550 nm) were determined at x100 magnification in 5 randomly chosen fields in duplicate dishes (1 mm²).

In Situ Labeling of DNA Fragments
Terminal deoxyribonucleotidyl transferase (TdT)–mediated dUTP nick end-labeling (TUNEL) was performed in cells plated on glass coverslips with the CardioTACS In Situ Apoptosis Detection kit (R&D Systems) according to the manufacturer’s instructions. For each staining procedure, control ARVMs were treated with TACS-nuclease working solution containing DNase and used as positive control. TUNEL-positive-staining cells were counted at x100 magnification in 5 randomly chosen fields (1 mm²) in triplicate plates under light microscopy and expressed as percentage of total cells.

Enzymatic Activities of the Upstream Caspase-8 and Caspase-9
Caspase-8 and -9 enzymatic activities were determined with Colorimetric Assay kits (R&D Systems). ARVMs from control, NE-treated, and NE+AT-treated dishes were washed with PBS, counted, trypsinized, and suspended in lysis solution. Caspase-8 and -9 specific substrates conjugated to the color reporter molecule p-nitroanilide were added to the cell lysates. After 2 hours of incubation at 37°C, p-nitroanilide was quantified spectrophotometrically at a wavelength of 405 nm in triplicate dishes (Microplate Reader EL 308, Bio-Tek). After correction for background activity and number of cells, enzyme activity was expressed as percent increase in activity over control.

Reverse Transcription–Polymerase Chain Reaction (RT-PCR) Studies
Total RNA was isolated from ARVMs exposed to NE (10 µmol/L), ISO (10 µmol/L), and ALB (100 µmol/L) alone or in combination with AT (10 µmol/L) for 12 hours with RNAzol (Tel-Test). Isolated RNA was reverse transcribed with a cDNA cycle kit (Invitrogen). PCR amplifications were done for Bax-, Bcl-2, and G3PDH with the following primers: Bax-, 5'-GTTTCATCCAGGATCGAGCAG-3' and 5'-CTTCCAGATGGTGAGCGAGG-3'; Bcl-2, 5'-AGCTGCACCTGACGCCCTT-3' and 5'-CAGCCA-GGAGAAATCAAACAGAGG-3'; G3PDH, 5'-ACCACAGTC-CATGCCATCAC-3' and 5'-TCCACCACCTGTTGCTGTA-3'. PCR products were analyzed by 1.5% agarose gel electrophoresis with a 100-bp ladder as size marker. All PCR reactions were done at least 3 times with different RNA preparations (n=3) and gave identical results. Photographic images were obtained, digitized, and quantified with SigmaGel (Jandel).

Statistical Analysis
Data are reported as mean±SEM. Student’s unpaired t test was used to compare groups. ANOVA with post hoc Scheffé test for multiple comparisons was performed to determine statistical significance of multiple treatments. Significance levels were established at a level of P<0.05.

	Results

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The present study was performed with Ca²⁺-tolerant and myocyte-enriched cardiac cultures. More than 99% of the cells stained positively with a myosin heavy chain–specific antibody, MF20,²⁵ as assessed in separate experiments.

AT, a ß₁-Selective AR Antagonist, Effectively Inhibits NE-Induced Apoptosis
TUNEL Staining and Annexin V/Propidium Iodide Staining
ARVMs were labeled with an in situ TUNEL assay (n=5). NE treatment (10 µmol/L) for 12 hours significantly increased the percentage of TUNEL-positive ARVMs compared with control cells (NE, 33±3% versus control, 3±1%; P<0.0001). Concomitant AT treatment (10 µmol/L) completely blocked the NE-induced increase in apoptotic cells (NE+AT, 4±2% versus control, 3±1%; P=0.98) (Figure 1). AT treatment alone did not affect the number of TUNEL-positive cells. AT treatment also decreased the percentage of NE-exposed ARVMs exhibiting an early apoptotic labeling pattern (annexin V–positive but propidium iodide–negative, An+/PI-: NE alone, 29±1% versus NE+AT, 5±1%; P<0.0001) as well as a late apoptotic (or necrotic) labeling pattern (annexin V– and propidium iodide–positive, An+/PI+: NE, 11±2% versus NE+AT, 2±1%; P<0.0001) to control levels (n=5) (Figure 2).

View larger version (77K): [in this window] [in a new window]   Figure 1. DNA strand breaks as assessed by TUNEL staining of ARVMs. ARVMs grown on coverslips were exposed to NE (10 µmol/L) alone or in presence of AT (10 µmol/L) for 12 hours and subjected to TUNEL staining. A, Mean percentages of TUNEL-positive ARVMs on coverslips. Data are mean±SEM of 5 experiments, each performed in triplicate. *P<0.0001 vs control (CTL); P<0.0001 vs NE. B, CTL ARVMs with rod-shaped morphology. C, NE-treated ARVMs with rounded morphology. D, NE+AT-treated ARVMs. Note preservation of rod-shaped morphology under NE treatment (B through D, bar=50 µm).

View larger version (30K): [in this window] [in a new window]   Figure 2. Annexin V/propidium iodide staining was performed to assess phosphatidylserine translocation from inner to outer cell surface. ARVMs grown on coverslips were exposed to NE (10 µmol/L) alone or in presence of AT (10 µmol/L) for 12 hours and subjected to annexin V/propidium iodide staining. A, Mean percentages of adherent cells with early apoptotic pattern of labeling (AN+/PI-) as well as late apoptotic pattern of labeling (An+/PI+). Data are mean±SEM of 5 experiments, each performed in duplicate. *P<0.0001 vs control (CTL); P<0.0001 vs NE. B, Early apoptotic pattern of annexin V/propidium iodide staining: An+/PI-. C, Late apoptotic pattern of annexin V/propidium iodide staining: An+/PI+ (bar=25 µm).

DNA Laddering
After 12 hours of treatment with NE (10 µmol/L), total genomic DNA from ARVMs was labeled with [³²P]dCTP by use of TdT (n=4). NE treatment for 12 hours markedly increased the intensity of 180- to 1000-bp fragments compared with control dishes. In addition, there was also increased background staining ("DNA smear") on NE treatment, which presumably represented NE-induced necrosis. Concomitant AT treatment clearly decreased the intensity to levels observed in control dishes, as assessed by visual inspection and densitometry (data not shown) (Figure 3A).

View larger version (26K): [in this window] [in a new window]   Figure 3. A, DNA laddering. Total genomic DNA was isolated, labeled with [32P]dCTP, and size-fractionated by electrophoresis. NE (10 µmol/L)–induced increase in DNA fragmentation is blocked by treatment with AT (10 µmol/L). CTL indicates control; M, 1-kb marker. B, Caspase-8 and -9 activities. After correction of background activity and number of cells, enzymatic activity was expressed as percent increase in activity over control cells. NE (10 µmol/L) induced a significant increase in caspase-9 activity, which was inhibited by treatment with AT (10 µmol/L). Caspase-8 was not activated in NE-treated cultures. Data are mean±SEM of 3 experiments, each performed in duplicate. *P<0.0001 vs CTL; P>0.70 vs CTL.

Caspase-8 and -9 Enzymatic Activity
Caspase-9 enzymatic activity was significantly increased by 200% over control dishes in NE-treated (10 µmol/L) ARVMs, whereas no caspase-8 enzymatic activity was detectable. AT treatment (10 µmol/L) effectively inhibited the increase in caspase-9 activity under NE treatment (Figure 3B).

Taken together, these data suggest that NE-induced apoptosis is mediated primarily by ß₁-selective adrenergic stimulation in ARVMs in vitro.

NE, ISO, and ALB Exhibit Distinct Dose-Response Curves as Assessed by TUNEL Staining
Because NE exerts only weak ß₂-adrenergic stimulation, additional studies were performed with increasingly ß₂-selective adrenergic agonists, including ISO (ß₁ß₂) and ALB (ß₁<ß₂). To rule out the possibility that NE elicits its effects partly via stimulation of AR, the AR antagonist prazosin was used in all subsequent experiments. Apoptosis dose-response curves were established for ARVMs exposed to increasing concentrations of NE, ISO, and ALB (1, 10, and 100 µmol/L) over 12 hours and subsequently subjected to TUNEL staining (n=3). NE and ISO significantly increased the percentages of apoptotic ARVMs at all determined concentrations compared with control cells (Figure 4). However, NE was more effective in inducing apoptosis at 10 and 100 µmol/L than ISO (at 100 µmol/L, NE>ISO>ALB, P<0.0001; at 10 µmol/L, NE>ISO>ALB, P<0.0001; at 1 µmol/L, NEISO>ALB, NE or ISO versus ALB, P<0.0001; NE versus ISO, P=0.62). Conversely, ALB induced only a relatively small percentage of apoptotic cells at the highest determined concentration (ALB at 100 µmol/L, 14±1%).

View larger version (12K): [in this window] [in a new window]   Figure 4. Apoptosis dose-response curves were established for ARVMs exposed to increasing concentrations of NE, ISO, and ALB (1, 10, and 100 µmol/L) over 12 hours and subsequently subjected to TUNEL staining. NE and ISO significantly increased percentages of apoptotic ARVMs at all determined concentrations compared with control cells. ALB induced only a small amount of apoptosis at highest concentration. Data are mean±SEM of 3 experiments, each performed in duplicate. *P<0.01 vs control (CTL).

The data presented suggest that increased ß₂-selective adrenergic agonism does not increase the percentage of ARVMs undergoing apoptosis but rather parallels a decrease in apoptosis.

ICI 118551, a Highly Selective ß₂AR Antagonist, Does Not Decrease NE-, ISO-, and ALB-Induced Apoptosis
To determine whether apoptotic death signaling by NE, ISO, and ALB would be mediated mainly by the ß₁AR, NE (10 µmol/L)–, ISO (10 µmol/L)–, and ALB (100 µmol/L)–exposed ARVMs were treated with PRO (ß₁=ß₂) (2 µmol/L), AT (ß₁>ß₂) (10 µmol/L), and ICI 118551 (ß₁<<ß₂) (0.1 µmol/L) for 12 hours and subsequently subjected to TUNEL staining (n=3). PRO and AT provided similar and complete protection in decreasing the percentages of apoptotic ARVMs (NE+AT versus NE+PRO versus control, P>0.60; ISO+AT versus ISO+PRO versus control, P>0.84; ALB+AT versus ALB+PRO versus control, P>0.80) (Figure 5). ALB-induced apoptosis at 100 µmol/L was completely blocked by AT, indicating a ß₁AR-mediated effect. Importantly, ICI 118551 did not decrease NE-, ISO-, and ALB-induced apoptosis (Figure 5). AT, PRO, and ICI 118551 treatment alone did not affect the number of TUNEL-positive cells.

View larger version (11K): [in this window] [in a new window]   Figure 5. ICI 118551 (0.1 µmol/L) did not decrease NE (10 µmol/L)–, ISO (10 µmol/L)–, and ALB (100 µmol/L)–induced apoptosis as assessed by TUNEL staining. PRO (2 µmol/L) and AT (10 µmol/L) provided complete protection in decreasing percentages of ARVMs undergoing apoptosis, whereas ICI 118551 did not affect number of TUNEL-positive cells. Data are mean±SEM of 3 experiments, each performed in duplicate. *P<0.01 vs control (CTL); P>0.80 vs CTL; P>0.90 vs NE, ISO, and ALB, respectively.

These data suggest that ß-adrenergic apoptotic death signaling is dissociated from ß₂ARs and selectively mediated by ß₁ARs.

NE-, ISO-, and ALB-Induced Changes in the Bcl-2–to-Bax Ratio Are Abolished by the ß₁-Selective AR Antagonist AT
Total RNA was isolated from ARVMs exposed to NE (10 µmol/L), ISO (10 µmol/L), and ALB (100 µmol/L) alone or treated with AT (10 µmol/L) for 12 hours and subjected to RT-PCR studies for Bax and Bcl-2 (n=3). NE, ISO, and ALB treatment increased the intensity of the signal corresponding to Bax and concomitantly decreased the signal corresponding to Bcl-2 compared with control (Figure 6). Consequently, the Bcl-2–to-Bax ratio was decreased compared with control ARVMs. AT treatment decreased the intensity of the signal corresponding to Bax and concomitantly increased the intensity of the signal corresponding to Bcl-2, thereby normalizing the Bcl-2–to-Bax ratio.

View larger version (39K): [in this window] [in a new window]   Figure 6. Bax- and Bcl-2 expression. Total RNA was isolated from ARVMs exposed to NE (10 µmol/L), ISO (10 µmol/L), and ALB (100 µmol/L) alone or treated with AT (10 µmol/L) for 12 hours and subjected to RT–PCR studies to determine Bcl-2–to-Bax ratio. Data are mean±SEM of 3 experiments. *P<0.0001 vs control (CTL); P>0.60 vs CTL.

These data strongly support the results obtained by the TUNEL technique. In particular, they indicate that the susceptibility to apoptotic stimuli is abnormally increased in ARVMs subjected to ß-adrenergic stimulation and that this abnormality is effectively prevented by treatment with AT, a ß₁-selective AR antagonist.

	Discussion

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The results of the present study demonstrate that ß₁-selective AR antagonism effectively inhibits NE-induced apoptosis in ARVMs. Furthermore, highly selective ß₂AR antagonism did not affect ßAR-mediated apoptosis. Thus, these observations strongly suggest that ß-adrenergic apoptotic death signaling is largely dissociated from ß₂ARs and is selectively mediated by ß₁ARs. After submission of our manuscript, similar findings were reported by Communal et al.¹⁷

Apoptosis, or programmed cell death, constitutes a key event in the pathogenesis of cardiac failure.¹⁵ Recent observations emphasize the fact that cardiomyocyte apoptosis is a critical event in the transition between compensatory cardiac hypertrophy and heart failure.²⁶ Several endogenously released excitotoxic substances have been implicated in the progressive deterioration of the failing heart, and catecholamines, released by enhanced compensatory sympathetic nerve activity, play a pivotal role. Communal et al¹¹ demonstrated that NE, acting via the ß-adrenergic pathway, induces cardiac apoptosis by phosphorylation of L-type Ca²⁺ channels through activation of protein kinase A (PKA), and more recently that ß₁ARs are involved in the process.¹⁷ Further evidence of apoptotic cardiomyocyte death by activation of the ßAR signaling pathway was reported from ISO-treated rat hearts²⁷ and transgenic mice overexpressing G_s²⁸ or G_q.²⁹

In the present study, a combination of techniques was used to assess apoptotic events, including staining procedures (in situ DNA end-labeling, annexin V/propidium iodide staining), DNA gel electrophoresis, determinations of enzymatic activities of the upstream caspases-8 and -9, and the expression ratio of the proapoptotic oncoprotein Bax and the antiapoptotic oncoprotein Bcl-2. The results of these methods were internally consistent, except that there was not a good correlation of in situ DNA end-labeling and Bcl-2–to-Bax ratio. Bcl-2–to-Bax ratio determines the susceptibility of myocytes to death after an apoptotic stimulus³⁰ but may not correlate linearly with the intensity of the apoptotic stimulus.³¹ DNA fragmentation (TUNEL staining) thus may not necessarily correlate with Bcl-2–to-Bax ratio. Importantly, however, in our experiments, ß₁-selective AR antagonism completely reversed ß-adrenergic signaling–induced changes in the Bcl-2–to-Bax ratio.

Our observations now support the concept that ß-adrenergic apoptotic cell death signaling is selectively mediated by ß₁ARs. The mechanisms involved, however, are not yet clear. Although transmembrane flux of Ca²⁺ represents an important commonality in the function of ßAR subtypes, they modulate cardiac function differently.⁸ Although both ß₁ARs and ß₂ARs increase contractile amplitude and hasten relaxation in rat and canine ventricular myocytes, several striking differences with respect to GTP-binding protein characteristics and signal transduction downstream from the ßAR have been revealed recently. In contrast to ß₁ARs, ß₂ARs exhibit a strong coupling to pertussis-toxin–sensitive G_i protein that can completely negate the ß₂AR G_s-mediated contractile, [Ca²⁺]_i, and I_Ca responses.⁶ Consistent with the recent report that ß₂ARs inhibit apoptosis via a G_i-coupled pathway,¹⁷ increased coupling to G_i protein by ß₂ARs has been shown to activate mitogen-activated protein kinase,³² which is known to confer antiapoptotic effects in cardiomyocytes.³³ Furthermore, ß₂AR subtype stimulation lacks phosphorylation of cytoplasmic PKA target proteins in rats and dogs, because ß₂AR-coupled cAMP/PKA signaling remains highly localized to subsarcolemmal microdomains in the vicinity of L-type Ca²⁺ channels.³ Finally, ß₂AR subtype stimulation reportedly has no measurable effects on diastolic Ca²⁺ and does not induce diastolic Ca²⁺ oscillations.⁸ Numerous studies have emphasized the important role of intracellular free Ca²⁺ on apoptosis.³⁴ Increased cytosolic Ca²⁺ affects mitochondrial membrane permeability and triggers the release of apoptogenic factors, including caspase-9 from damaged mitochondria.³⁵ The observed activation of caspase-9 but not caspase-8 is of interest. Two major pathways, one involving cytochrome c, Apaf-1, and caspase-9 of mitochondria and the other mediated through CD95 receptor and caspase-8, have been identified in mammalian cells.³⁶ Our results indicate a pivotal role of the mitochondria in catecholamine-induced apoptosis in cardiomyocytes.

The potential role of ß₂AR stimulation for improving cardiac performance in the failing heart has received considerable attention.^{18 19} In transgenic mice with overexpression of G_q, an experimental model for hypertrophy and heart failure, overexpression of ß₂ARs at levels that preserve the specificity and fidelity of the ß₂AR signaling pathway (30-fold overexpression compared with wild-type) extended favorable effects on hemodynamic parameters, expression of fetal genes, and hypertrophy.¹⁹ This implies that ßAR signaling does not exclusively mediate deleterious effects but may include beneficial effects in the compromised heart. The reported findings in transgenic mice overexpressing G_q/ß₂ARs¹⁹ and ß₁AR³⁷ are consistent with our observations and suggest the potential therapeutic role of a combined pharmacological treatment, including ß₁AR antagonism and ß₂AR agonism in the failing heart. ß₁AR antagonism has been shown to improve outcome in congestive heart failure,³⁸ and interestingly, it enhances the ß₂AR-mediated inotropic response to catecholamines.³⁹

Our experiments, however, must be interpreted with caution. First, significant species differences, in particular with respect to G-protein coupling, may influence the receptor subtype–specific response to stimuli for apoptosis. Second, although AT has been shown to selectively inhibit the sympathetic response in murine wild-type hearts but not in ß₂AR-overexpressing hearts of mice,²² the pharmacological selectivities for ßAR subtypes of the agonists and antagonists used in the present study are limited. Therefore, we cannot exclude the possibility that a small amount of apoptosis might have occurred by ß₂AR stimulation or that other putative ßARs might be involved. Also, the apoptotic effects on myocytes were assessed by potent short-term ßAR stimulation, which might not reflect ßAR-mediated long-term effects on apoptotic cell death.

In conclusion, the experimental results of the present study provide evidence that ß-adrenergic apoptotic death signaling is selectively mediated by the ß₁AR in ARVMs in vitro. The observed dissociation of the ß₂AR from apoptotic death signaling supports the use of ß₂AR stimulation as a potential therapy to improve inotropic and lusitropic function in the failing heart.

Received November 22, 1999; revision received February 14, 2000; accepted February 21, 2000.

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