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(Circulation. 2000;102:2249.)
© 2000 American Heart Association, Inc.

Basic Science Reports

Chronic -Adrenergic Receptor Stimulation Modulates the Contractile Phenotype of Cardiac Myocytes In Vitro

Naoya Satoh, BS; Thomas M. Suter, MD; Ronglih Liao, PhD; Wilson S. Colucci, MD

From the Cardiovascular Section, Boston University Medical Center, Myocardial Biology Unit and Cardiac Muscle Research Laboratory, Boston University School of Medicine, Boston, Mass.

Correspondence to Dr Wilson S. Colucci, Cardiovascular Section, Boston University Medical Center, 88 E Newton St, Boston, MA 02118. E-mail wilson.colucci{at}bmc.org

	Abstract

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Background—Heart failure is characterized by contractile dysfunction of the myocardium and elevated sympathetic activity. We tested the hypothesis that chronic -adrenergic (-ADR) stimulation modifies the molecular and contractile phenotype of cardiac myocytes.

Methods and Results—Adult rat ventricular myocytes in culture were exposed to -ADR stimulation (norepinephrine + propranolol) for 48 hours. -ADR stimulation decreased the mRNAs for sarcoplasmic reticulum Ca²⁺-ATPase and Ca²⁺ release channel by 56% and 52%, respectively, and increased mRNA and protein for the Na⁺-Ca²⁺ exchanger by 70% and 39%, respectively. After washout of the -ADR agonist, simultaneous measurement of [Ca²⁺]_i transients with fura 2 and myocyte shortening by video edge-detection showed that [Ca²⁺]_i amplitude and myocyte shortening were decreased in -ADR–treated myocytes, and the time to peak and time from peak to 80% decline of both [Ca²⁺]_i and myocyte shortening were increased. The concentration-response curve for myocyte shortening by the Na⁺ channel activator veratridine was shifted leftward in -ADR–stimulated myocytes (EC₅₀, 21.6±4.6 versus 105.8±10.5 nmol/L, P<0.001).

Conclusions—Chronic -ADR stimulation of cardiac myocytes causes decreases in the expression of sarcoplasmic reticulum Ca²⁺-ATPase and the Ca²⁺ release channel that are associated with decreases in [Ca²⁺]_i and contractility. -ADR stimulation simultaneously increases Na⁺-Ca²⁺ exchanger expression, thereby increasing sensitivity to intracellular Na⁺.

Key Words: myocytes • calcium • sarcoplasmic reticulum • ion channels • receptors, adrenergic, alpha

	Introduction

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Deranged Ca²⁺ homeostasis due to alterations in Ca²⁺-handling proteins may play an important role in the pathogenesis of myocardial dysfunction in patients with heart failure.^{1 2} In failing human myocardium, sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2) is decreased,^{1 3} whereas the Na⁺-Ca²⁺ exchanger (NCX) is increased.^{4 5 6} Expression of the sarcoplasmic reticulum Ca²⁺ release channel (CRC) may be depressed, although this finding appears to be inconsistent.^{7 8 9} The reciprocal changes in SERCA2 and NCX expression are of particular interest because increased NCX activity could theoretically compensate for depressed SERCA2 function, particularly with regard to diastolic Ca²⁺ removal.^{4 10} In support of this thesis, Hasenfuss et al¹¹ found that, in failing human myocardium with reduced expression of SERCA2, diastolic function was preserved if NCX was increased but was impaired if NCX was not increased. The mechanism responsible for regulation of myocyte SERCA2, NCX, and CRC in failing myocardium is not known.

Heart failure is characterized by elevated sympathetic activity,¹² which might, over time, impair myocardial function by several mechanisms, including Ca²⁺ overload, hypoxia, increased sarcolemmal membrane permeability, and myocyte death.^{13 14} Most attention has been focused on the role of the ß-ADR in mediating these and other adverse effects of sympathetic stimulation. However, -ADRs are expressed in the myocardium in most species,^{15 16} including humans,¹⁷ where they exert a modest effect on contractility.¹⁸

Chronic -ADR stimulation might modulate the contractile phenotype of the myocardium. In vitro, -ADR stimulation has marked effects on myocyte phenotype, including the stimulation of cell hypertrophy,^{19 20} induction of fetal genes,²¹ and the elaboration of peptide growth factors²² that can stimulate fetal gene expression.²³ Fetal gene expression is often associated with reciprocal changes in the expression of adult genes involved in calcium homeostasis, such as SERCA2.¹ Accordingly, our goal was to use adult rat ventricular myocytes (ARVMs) as an in vitro system to test the hypothesis that chronic -ADR stimulation regulates the expression of calcium-handling proteins and thereby modulates the contractile phenotype of the myocyte.

	Methods

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Isolation and Culture of ARVMs
Calcium-tolerant ARVMs were isolated as previously described.¹⁴ In brief, the hearts of adult male Wistar rats were perfused retrogradely for 4 sequential periods as follows: (1) with Tyrode’s solution (in mmol/L: NaCl 137, KCl 5.4, CaCl₂ 1.8, MgCl₂ 0.5, HEPES 10, and glucose 10, pH 7.4) for 5 minutes, (2) with nominally Ca²⁺-free Tyrode’s solution (in mmol/L: NaCl 135, KCl 4, MgCl₂ 1, HEPES 10, NaH₂PO₄ 0.33, and glucose 10, pH 7.2) for 6 minutes, (3) with nominally Ca²⁺-free Tyrode’s solution containing collagenase A (0.08%, Boehringer Mannheim) and protease (0.02%, Sigma) for 6 to 12 minutes, and (4) with KB solution (in mmol/L: KOH 85, KCl 30, KH₂PO₄ 30, MgSO₄ 3, EGTA 0.5, HEPES 10, L-glutamic acid 50, taurine 20, and glucose 10, pH 7.4) for 5 minutes. All solutions were aerated with 100% oxygen and kept at 37°C. The left ventricle was cut into several pieces and shaken to facilitate cell dispersion. The cells were suspended in DMEM and layered over 60 mg/mL BSA (Sigma) to separate ventricular myocytes from nonmyocytes. The cells were plated onto laminin-coated culture dishes (Fisher) or glass coverslips (Fisher) and maintained in ACCIT medium (BSA 2 mg/mL, L-carnitine 2 mmol/L, creatine 5 mmol/L, insulin 20 IU/L, taurine 5 mmol/L, penicillin 100 IU/mL, and streptomycin 100 µg/mL in DMEM) for 24 hours before the addition of agonist.

Myocyte Treatments
For -ADR stimulation, l-norepinephrine (10 µmol/L, Sigma) and propranolol (2 µmol/L, Sigma) were added to the culture medium for 48 hours. The medium was changed at 24 hours of the treatment. In some experiments, prazosin (3 µmol/L, Sigma), U73122 (3 µmol/L, Alexis), or staurosporine (100 nmol/L, Sigma) was added 30 minutes before the agonist.

RNA Preparation and Northern Blot Analysis
Total RNA from ARVMs was prepared and separated as previously described²⁴ by a modification of the protocol of Chomczynski and Sacchi.²⁵ RNA was transferred to nylon membranes (DuPont-NEN) by capillary transfer and cross-linked by UV irradiation. The blot was successively hybridized with a 500-bp rat cDNA for NCX, a 1300-bp rat cDNA for SERCA2, and a 600-bp rabbit cDNA for CRC at 42°C overnight. The cDNAs were labeled with [³²P]dCTP (Amersham) to a specific activity of 1x10^-6 to 2x10^-6 cpm/ng cDNA by the random hexamer priming method. Blots hybridized with NCX and CRC cDNAs were washed twice (15 minutes, room temperature) with 2xSSC (1xSSC: 150 mmol/L NaCl, 15 mmol/L trisodium citrate, pH 7.0)/0.1% SDS and once (5 to 20 minutes, 45°C) with 0.5xSSC/0.1% SDS. Blots hybridized with SERCA2 cDNA were washed twice (15 minutes, room temperature) with 2xSSC/0.1% SDS and twice (15 minutes, 60°C) with 0.2xSSC/0.1% SDS. All blots were rehybridized with synthetic oligonucleotide complementary to 18S ribosomal RNA. Blots were exposed to Kodak X-OMAT films with 2 intensifying screens at -80°C. The signals were quantified by densitometric analysis (GS-700, BioRad).

Protein Preparation and Immunoblotting
Myocytes were scraped off into lysis buffer (in mmol/L: Tris-HCl 20, EDTA 1, dithiothreitol 1, leupeptin 0.1, phenylmethylsulfonyl fluoride 0.2, pH 7.4). Cell suspension was sonicated 2 times for 15 seconds with a 15-second interval. The cell suspension was then homogenized for 1 minute with a glass-glass homogenizer. The suspension was centrifuged at 100 000g for 30 minutes. The supernatant was discarded, and the pellet was resuspended in 5-fold volume of lysis buffer. Samples of 30 µg protein were denatured by heating to 95°C in sample buffer (62.5 mmol/L Tris-HCl, 2% SDS, 25% glycerol, 0.01% bromphenol blue) and subjected to SDS-PAGE (7.5% running gel) electrophoresis. Proteins were transferred to nitrocellulose membranes (0.2 µm, Schleicher and Schuell) by semidry electrophoretic blotting (PowerPac 3000, BioRad) at 5 mA/cm² for 1 hour with transfer buffer (25 mmol/L Tris-HCl [pH 8.3], 192 mmol/L glycine, 20% [vol/vol] methanol). The membrane was stained with Ponceau S (Sigma) to confirm equal loading of the samples. After destaining, the membrane was incubated for 2 hours in PBS buffer (in mmol/L: NaCl 137, KCl 2.7, KH₂PO₄ 100, NaH₂PO₄ 10.4, pH 7.4) with 5% nonfat milk and 0.05% Tween 20. The membrane was then incubated overnight with a rabbit antiserum raised against the canine NCX (Swant) diluted 1:200 in PBS buffer containing 5% nonfat milk. After washings with PBS buffer containing 0.05% Tween 20, the membrane was incubated for 1 hour with a 1:10 000 dilution of peroxidase-conjugated goat secondary antibody raised against rabbit IgG for immunodetection. After repeated washes, detection was performed with an enhanced chemiluminescence kit (SuperSignal, Pierce). The membrane was then exposed to Kodak X-OMAT film. The signals were quantified by densitometric analysis (GS-700, BioRad).

Contractility and [Ca²⁺]_i
Contractile properties and [Ca²⁺]_i of ARVMs were measured as recently described.²⁶ Glass coverslips with attached myocytes were incubated in Tyrode’s buffer (in mmol/L: NaCl 137, KCl 5.4, CaCl₂ 1.2, MgCl₂ 0.5, HEPES 10, and glucose 10, pH 7.4, 37°C) containing 1 µmol/L of membrane-permeant fura 2-AM (Molecular Probes) for 10 minutes and rinsed with Tyrode’s buffer containing 500 µmol/L of probenecid to prevent leakage of fura 2. The myocytes were field-stimulated at 2 Hz by a stimulator (Grass Instruments). The myocytes were superfused with Tyrode’s buffer for 1 hour before they were studied to (1) wash out the agonist and inhibitors and (2) allow for deesterification of the fura 2-AM. Myocyte length was monitored from a red-light bright-field image (650-nm long-pass filter). Cell length was measured by a video edge-detection system (IonWizard software, IonOptix). The epifluorescence signal was measured by a photomultiplier. [Ca²⁺]_i was calibrated according to the protocol of in vitro calibration.²⁷

Na⁺,K⁺-ATPase Activity
Na⁺,K⁺-ATPase was prepared as described by Watanabe et al.²⁸ Na⁺,K⁺-ATPase assay was carried out in standard buffer (in mmol/L: ATP 5, NaCl 100, KCl 10, MgCl₂ 5, Tris-HCl 40, EDTA 1, NaN₃ 15, pH 7.4, 37°C). Na⁺,K⁺-ATPase specific activity was defined as 1 µmol of inorganic phosphate liberated per mg protein per hour, after subtraction of inorganic phosphate liberated in the presence of 1 mmol/L ouabain.

Statistical Analysis
The results are presented as mean±SEM. When 2 groups were compared, Student’s t test was used. When more than 2 groups were compared, 1-way ANOVA was used, and post hoc multiple comparisons were performed by Bonferroni’s test. Differences were considered significant at a value of P<0.05.

	Results

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Effect of -ADR Stimulation on Myocyte Viability
The viability of myocytes was assessed by counting the number of rod-shaped myocytes. After -ADR stimulation for 48 hours, myocyte viability was 86±7% and 88±6% of the pretreatment number in control and -AR treated plates, respectively (n=5 plates each, P=NS).

-ADR Stimulation Regulates Ca²⁺-Handling Protein mRNA Levels
-ADR stimulation for 48 hours decreased the mRNA levels for SERCA2 (-56±4%; n=5; P<0.001 versus control myocytes) and CRC (-52±9%; n=5; P<0.01) as assessed by Northern blot analysis (Figure 1). In contrast, the level of mRNA for NCX increased (+70±8%; n=5; P<0.01).

View larger version (26K): [in this window] [in a new window]   Figure 1. A, Representative Northern blot analysis of NCX, SERCA2, and CRC in control and -ADR–stimulated (-ADR) ARVMs. cDNAs for NCX, SERCA, and sarcoplasmic reticulum CRC hybridized specifically at 7.2, 4.3, and 16.0 kb, respectively. B, Average changes in mRNA levels for NCX, SERCA2, and CRC. mRNA levels are related to levels of 18S rRNA. Data are mean±SEM. n=5 in each group. **P<0.01, ***P<0.001 vs control mRNA levels.

Pretreatment with the ₁-ADR antagonist prazosin, the phospholipase C inhibitor U73122, or the protein kinase C inhibitor staurosporine had no effect on basal NCX mRNA level, but each completely inhibited the increase caused by -ADR stimulation (Figure 2).

View larger version (18K): [in this window] [in a new window]   Figure 2. Effects of (A) prazosin, (B) U73122, or (C) staurosporine (Stauro) on increase in NCX mRNA level by -ADR stimulation (-ADR) in ARVMs. Antagonists/inhibitors were added to culture medium 30 minutes before application of -ADR agonist. Data are mean±SEM. n=3 to 5. **P<0.01, ***P<0.001 vs control myocytes (no -ADR agonist, no antagonist/inhibitors); ##P<0.01, ###P<0.001 vs myocytes treated with -ADR agonist alone.

Effects of -ADR Stimulation on Myocyte [Ca]_i and Contraction
Myocyte [Ca²⁺]_i and length were measured after -ADR washout (Table 1). In -ADR–stimulated myocytes, systolic [Ca²⁺]_i was decreased by 40% (P<0.001; n=20), diastolic [Ca²⁺]_i was decreased by 31% (P<0.001; n=20), and [Ca²⁺]_i amplitude was decreased by 47% (P<0.001; n=20) compared with control myocytes. The time to peak of [Ca²⁺]_i and the time from peak to 80% decline of [Ca²⁺]_i were increased by 39% (P<0.001) and 42% (P<0.001), respectively, in -ADR–stimulated myocytes.

View this table: [in this window] [in a new window]   Table 1. [Ca2+]i and Myocyte Length in Control and -ADR–Stimulated Myocytes

Diastolic and systolic myocyte lengths at rest were unchanged by chronic -ADR stimulation. Myocyte shortening was decreased 35% (P<0.01) in -ADR–stimulated myocytes, the time to peak myocyte shortening was increased 13% (P<0.05), and the time from peak to 80% decline of myocyte shortening was increased 83% (P<0.001).

Effects of -ADR Stimulation on NCX Protein Expression
NCX protein levels were measured by Western blot with a specific polyclonal antibody.²⁹ In myocytes treated with -ADR stimulation for 48 hours, NCX protein level was increased 39±8% (P<0.05 versus control myocytes; n=6) (Figure 3).

View larger version (18K): [in this window] [in a new window]   Figure 3. A, Representative Western blot analysis of NCX in control and -ADR–stimulated (-ADR) ARVMs. Immunochemical detection revealed 2 protein bands at 120 and 70 kDa in control and -ADR–stimulated (-ADR) ARVMs. Because both bands are associated with NCX, NCX protein level was quantified by adding up densitometric readings of both bands. B, Average changes in protein levels for NCX. Data are mean±SEM. n=5 in each group. *P<0.05 vs control protein level.

Effect of -ADR Stimulation on the Contractile Response to Veratridine
To assess the physiological significance of increased NCX expression, the contractile response to increasing intracellular Na⁺ was assessed by use of the Na⁺ channel activator veratridine.³⁰ Myocytes were superfused with Tyrode’s buffer for 1 hour to wash out the -ADR agonist before the response to veratridine was tested. In cells treated with -ADR stimulation for 48 hours, basal myocyte shortening was decreased by 29% (P<0.01; n=7), as was the maximum response to veratridine, which was decreased by 25% (P<0.01; n=7) (Figure 4A). Normalization of the veratridine concentration-response relationships to their respective maximal effects (Figure 4B) demonstrated that chronic -ADR stimulation caused a leftward shift such that the median effective concentration (EC₅₀) for veratridine was decreased from 105.8±10.5 to 21.6±4.6 nmol/L (P<0.001; n=7), indicative of increased sensitivity to intracellular Na⁺. Increased myocyte sensitivity to intracellular sodium might also reflect a decrease in the activity of Na⁺,K⁺-ATPase. However, Na⁺,K⁺-ATPase activity was not affected by chronic -ADR stimulation (Table 2).

View larger version (18K): [in this window] [in a new window]   Figure 4. A, Concentration-response curves for effects of veratridine on myocyte shortening in control and -ADR–stimulated (-ADR) ARVMs. Experiments were done 1 hour after washout of -ADR agonist to eliminate its acute effects. B, Concentration-response curves for effect of veratridine on myocyte shortening were normalized by their respective maximum effects. EC50 for veratridine response were 105.8±10.5 nmol/L in control myocytes and 21.6±4.6 nmol/L in -ADR–stimulated myocytes (P<0.001). Data are mean±SEM. n=7 in each group.

View this table: [in this window] [in a new window]   Table 2. Sarcolemmal Na+,K+-ATPase Activity in Control and -ADR–Stimulated Myocytes

	Discussion

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This study demonstrates that in ARVMs in vitro, chronic -ADR stimulation decreased the expression of SERCA2 and CRC and increased the expression of the NCX. These changes in gene expression were associated with changes in the amplitude and kinetics of [Ca²⁺]_i transients and myocyte shortening and with increased sensitivity to intracellular Na⁺.

Expression of a Fetal Gene Program
SERCA2, CRC, and NCX are developmentally regulated genes in the myocardium. The expression of SERCA2 and CRC increases with maturation,^{31 32} whereas the expression of NCX decreases.³³ Several growth factors that induce the expression of fetal genes cause reciprocal decreases in the expression of SERCA2 and CRC in cardiac myocytes.^{23 24} Because -ADR stimulation is known to induce the expression of fetal genes (eg, atrial natriuretic peptide, skeletal -actin) in cardiac myocytes,³⁴ it is not surprising that -ADR stimulation decreased the expression of SERCA2 and CRC. Likewise, although less is known about the regulation of NCX in cardiac myocytes, the observed increase in NCX is consistent with reexpression of a fetal gene program. In this regard, our findings are similar to those of Reinecke et al.³⁵ The ability of chronic -ADR stimulation to increase NCX mRNA expression was abolished by prazosin, an ₁-ADR antagonist; U-73122, an inhibitor of phospholipase C; and staurosporine, an inhibitor of protein kinase C, suggesting that the -ADR/phospholipase C/protein kinase C pathway mediates this effect.

Physiological Effects of Chronic -ADR Stimulation
The decreases in [Ca²⁺]_i amplitude and contractility with chronic -ADR stimulation are consistent with the observed decreases in the expression of SERCA2 and CRC, proteins that play important roles in providing Ca²⁺ for contraction. In cardiac myocytes, the acute effect of -ADR stimulation is to increase the amplitude of contraction.^{15 36} The chronic effects of -ADR stimulation are directionally opposite those observed with acute stimulation and thus cannot be attributed to the acute effect of -ADR stimulation.

Increased Contractile Sensitivity to Veratridine
To assess the physiological significance of increased NCX expression, we determined the contractile response to the Na⁺ channel activator veratridine. Veratridine increases intracellular Na⁺, which, in turn, activates the NCX, thereby increasing Ca²⁺ influx and contractility.³⁷ Veratridine increased myocyte shortening in a concentration-dependent manner in both control and -ADR–stimulated myocytes. In -ADR–stimulated myocytes, the maximum response to veratridine was decreased, whereas the concentration-response relationship was shifted leftward. The decrease in the maximum response to veratridine may reflect the reduced availability of intracellular Ca²⁺ as a result of decreased expression of SERCA2 and CRC. It is also possible that the reduced contractile response in -ADR–stimulated cells reflects changes in the expression of contractile proteins.³⁸ The increased sensitivity to veratridine in -ADR–stimulated myocytes is consistent with increased influx of extracellular Ca²⁺ via the NCX. Thus, the response to veratridine suggests that the increase in NCX protein is associated with increased NCX activity. An increase in NCX expression might serve to support cardiac contractility by increasing the responsiveness to Na⁺. This view is supported by the demonstration that the contractile response to the Na⁺ channel agonist BDF9148 is increased in atria from transgenic mice with overexpression of NCX.³⁹

Increased responsiveness to veratridine might also reflect an increase in intracellular Na⁺ due to increased Na⁺ influx and/or decreased efflux. However, chronic -ADR stimulation had no effect on the activity of Na⁺,K⁺-ATPase, which is the dominant protein responsible for pumping Na⁺ out of myocytes.

Possible Implications for Myocardial Failure
The changes in the expression of calcium handling proteins observed with chronic -ADR stimulation are similar to those observed in the failing human myocardium.^{1 3 4 7} Of note, increased expression of the NCX in failing human myocardium is associated with an increased contractile response to the Na⁺ channel agonist BDF9148.⁵ Likewise, the changes in [Ca²⁺]_i observed after chronic -ADR stimulation are generally similar to those observed in ventricular myocytes isolated from patients with severe heart failure, which exhibit a decrease in [Ca²⁺]_i amplitude and a slower decay in diastolic [Ca²⁺]_i than nonfailing myocytes.^{2 40 41} The contractile phenotype of failing myocytes probably reflects the net influence of multiple factors, such as -ADR stimulation, endothelin, inflammatory cytokines, and reactive oxygen species, which can affect the expression of calcium-handling proteins.⁴² In addition, chronic -ADR stimulation may regulate the expression of -ADR on myocytes,⁴³ which may further modify the responses to subsequent -ADR stimulation. Of note, we have found that chronic ß-adrenergic receptor stimulation causes apoptosis¹⁴ but has no effect on the expression of NCX, CRC, or SERCA2 mRNA or basal contractile phenotype (unpublished observations). The present experiments thus demonstrate that chronic stimulation of myocyte -ADR by adrenergic overactivity could contribute to the abnormal contractile phenotype observed in heart failure.

	Acknowledgments

 
This study was supported in part by grants HL-42539 and HL-61639 (Dr Colucci) from the NIH. We are grateful to Dr K.D. Philipson for providing the rat NCX cDNA, to Dr J. Lytton for providing the rat SERCA cDNA, and to Dr A.R. Marks for providing the rabbit sarcoplasmic reticulum CRC cDNA. We appreciate the technical assistance of D.L.F. Chang.

Received April 7, 2000; revision received June 5, 2000; accepted June 8, 2000.

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