Involvement of β³-Adrenoceptor in Altered β-Adrenergic Response in Senescent Heart: Role of Nitric Oxide Synthase 1–derived Nitric Oxide

This study, including care of the animals involved, was conducted according to the official edict presented by the French Ministry of Agriculture (A5550-01, Paris, France) and the recommendations of the Declaration of Helsinki. Therefore, these experiments were conducted in authorized laboratories and under the supervision of an authorized researcher for each institution (B.R., C.H., and R.A. for EA 3975, INSERM U689, and UMR-INSERM 771-CNRS 6214, respectively). Young adult (3 months) and senescent male Wistar rats (24 months) (Charles River, Saint Germain sur l'Arbresle, France), were used.

Echocardiography was performed on anesthetized rats (1–2% isoflurane) using a General Electric Vivid 7 instrument (Aulnay-sous-Bois, France) equipped with an 8- to 14-MHz linear transducer. Data were transferred on-line to a computer for analysis (EchoPAC PC version 2.0.x; General Electric). Left ventricular diameter was measured in the parasternal long-axis and short-axis views in M mode. Left ventricular ejection fraction and left ventricular fractional shortening were measured using a modified version of Simpson monoplane analysis.7,18Left ventricular diastolic parameters were derived from pulsed-wave spectral Doppler mitral flow and from pulsed-wave spectral mitral tissue Doppler imaging from apical view, with the sample volume paced at the lateral corner of the mitral annulus as reported previously.7,18,19Left ventricular systolic function was evaluated using the left ventricular diameter, left ventricular ejection fraction, and left ventricular fractional shortening. Left ventricular diastolic function was investigated using peak velocity of early (E) and late (A) filling waves and E/A ratio.7,18,19In addition, isovolumic relaxation time and deceleration time of the E wave were derived from pulsed-wave mitral Doppler spectra and the peak early diastolic velocity Ea from pulsed-wave spectral mitral tissue Doppler imaging.18,19The left ventricular end-diastolic pressure was measured using the E/Ea ratio.7,18,19Dobutamine (4 μg/kg) was administrated intraperitoneally, and measurements were performed when the increase in heart rate was stabilized.7,18 

Papillary muscle mechanics were studied in Krebs-Henseleit bicarbonate buffer solution as described previously.1,2,7,18,20After brief anesthesia with sodium pentobarbital, the heart was quickly removed. The whole heart and the left ventricle were dissected and weighed, and the left ventricular papillary muscles were carefully excised and suspended vertically in a 200-ml jacketed reservoir with Krebs-Henseleit bicarbonate buffer solution (118 mm NaCl, 4.7 mm KCl, 1.2 mm MgSO⁴, 1.1 mm KH²PO⁴, 25 mm NaHCO³, 2.5 mm CaCl², and 4.5 mm glucose) and maintained at 29°C with a thermostatic water circulator. Preparations were field stimulated at 12 pulses/min with 5-ms rectangular wave pulses set just above threshold. The bathing solution was bubbled with 95% O²and 5% CO², resulting in a pH of 7.40. After a 60-min stabilization period with the initial muscle length at the apex of the length–active isometric tension curve, papillary muscles recovered their optimal mechanical performance. The extracellular concentration of Ca²⁺was decreased from 2.5 to 0.5 mm because rat myocardial contractility is nearly maximal at 2.5 mm.18,20,21Conventional mechanical parameters at the initial muscle length at the apex of the length–active isometric tension curve were calculated from three twitches. The first twitch was isotonic and was loaded with the preload corresponding to the initial muscle length at the apex of the length–active isometric tension curve. The second twitch was abruptly clamped to zero load just after the electrical stimulus with a critical damping. The third twitch was fully isometric at the initial muscle length at the apex of the length–active isometric tension curve (L^max). We determined the maximum unloaded shortening velocity using the zero-load technique, and time to peak shortening of the twitch with preload only. In addition, the maximum isometric active force normalized per cross-sectional area and the time to peak force were recorded from the isometric twitch. At the end of the study, the muscle cross-sectional area was calculated from the length and weight of papillary muscle, assuming a density of 1.

β-Adrenoceptor stimulation was induced by cumulative concentrations of isoproterenol (10⁻⁸to 10⁻⁴m), a nonselective β-adrenoceptor agonist, in the presence of phentolamine (10⁻⁶m), a specific α¹-adrenoceptor antagonist.7,18 

To assess the role of the β³-adrenoceptor, we studied additional groups exposed to cumulative concentrations of BRL 37344 (10⁻⁸to 10⁻⁵m),22a specific β³-adrenoceptor agonist, in the presence of nadolol (10⁻⁵m), a specific β¹- and β²-adrenoceptor antagonist.22The effect of β³-adrenoceptor stimulation on the cyclic adenosine monophosphate resulting from the β¹- and β²-adrenoceptor stimulation was studied using dibutyryl-cyclic adenosine monophosphate (5.10⁻⁴m), a fat-soluble and diffusible analog of cyclic adenosine monophosphate resistant to hydrolysis in the intracellular involvement,7,18in the presence of nadolol (10⁻⁵m) and in the presence or not in the presence of BRL 37344 (10⁻⁵m).

To assess the NOS isoform involved in the β³-adrenoceptor pathway, we studied additional groups exposed to N  ^G-nitro-l-arginine methylester (l-NAME; 10⁻⁵m), an unspecific NOS inhibitor; to vinyl-l-N -5(1-imino-3-butenyl)-l-ornithine (l-VNIO; 10⁻⁴m), a specific NOS1 inhibitor; or to 1400W (10⁻⁴m), a specific NOS2 inhibitor, as reported previously.7,22 

The total volume of added drugs did not exceed 2% of the bath volume. All drugs were purchased from Sigma Chemical (L'Isle d'Abeau Chesne, France), except l-VNIO, which was purchased from Coger (Paris, France), and BRL 37344, which was purchased from Tocris Biosciences (Bristol, United Kingdom).

Detection of nitric oxide production was performed both in vivo  and in vitro  using the technique with Fe²⁺diethyldithiocarbamate (Sigma Chemical) as the spin trap as previously described.23To measure nitrite oxide production in vivo , Fe²⁺diethyldithiocarbamate was injected intraperitoneally (400 mg/kg) with or without injection of intraperitoneal dobutamine and FeSO⁴-7H²O (40 mg/kg) and citrate (200 mg/kg) by subcutaneous injection on the neck of adult or senescent rats. After 30 min, rats were killed to harvest the left ventricle from the heart to measure nitric oxide. In another set of experiments, in vitro , small pieces of left ventricular myocardium from adult and senescent rats were placed in 24-well clusters filled with 250 μl Krebs solution containing phentolamine (10⁻⁶m) with or without isoproterenol (10⁻⁶m) in presence or not in the presence of NOS inhibitors: l-NAME (100 μm), 1400W (100 μm), or l-VNIO (100 μm). The left ventricular myocardium was treated with 250 μl of the colloid Fe(Fe²⁺diethyldithiocarbamate)²and incubated at 37°C for 1 h. All nitric oxide measures were performed on a tabletop x-band spectrometer miniscope (Magnettech, Berlin, Germany). Recordings were made at 77°K using a Dewar flask. Instrument settings were 10 mW of microwave power, 1 mT of amplitude modulation, 100 kHz of modulation frequency, 60 s of sweep time, and 10 scans.

Staining and imaging of NOS1 and NOS2 were investigated by confocal microscopy. Pieces of left ventricular myocardium were frozen and cut into 7-μm sections. Fixed sections were incubated (2 h at room temperature) in a blocking buffer (5% nonfat dry milk in phosphate-buffered saline). Tissue sections were then incubated overnight (4°C) with monoclonal murine anti-NOS2 (1:100; Transduction Laboratories, Heidelberg, Germany) or anti-NOS1 (1:100; Transduction Laboratories) antibodies. Three washes were followed by incubation (1 h, 37°C) with secondary murine and rabbit, respectively, fluorescent Alexa fluor-488-labeled antibody (1:100; Invitrogen Molecular Probes, Leiden, The Netherlands). Slides were examined with an Olympus light microscope Fluoview FU 300 Laser Scanning Confocal Imaging System (Olympus, Paris, France) equipped with an argon ion laser (EM 488 nm). Pictures were taken with a ×10 objective (water immersion). The laser was adjusted in the green fluorescent mode. Z series were collected in 1-μm steps, and final images were obtained after stacking.

Western Blots were performed on left ventricular homogenates with specific antibodies to measure protein expression of β¹- and β³-adrenoceptors, as described previously.7,18Briefly, cardiomyocytes were homogenized in Triton X-100 buffer (1% Triton X-100 with 50 mm Tris-HCl [pH 7.4], 100 mm NaCl, 50 mm NaF, 5 mm EDTA, 40 mm β-glycerophosphate, 0.2 mm orthovanadate, 0.1 mm leupeptin, and 0.001 mm aprotinin) for 1 h at 4°C. After centrifuging at 15,000g  for 15 min at 4°C, supernatant protein concentrations were measured using the BCA protein assay kit (Perbio Science, Brebières, France). Proteins were prepared as previously described,7,18and 50 μg protein per lane was immunoblotted using anti–β¹-adrenoceptor (1:1,000; Affinity Bioreagents, Saint Quentin en Yvelines, France) and goat polyclonal anti–β³-adrenoceptor (1:1,000; Santa Cruz Biotechnology, Le Perray en Yvelines, France). All the Western blot experiments were quantified using normalization, including a standardization of the different gels by loading a reference sample on every gel and checking that a similar total amount of protein was loaded by measurement of total protein level present on the membrane colored by S-Ponceau. The S-Ponceau staining enabled us to verify that equal amounts of protein were loaded. Accordingly, all of the results were normalized with a link (actin) and the amounts of protein transferred on the membrane. A control by performing a Western blot using a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase, was performed and validated that there was no variation in protein gel loading in our hands.

Data are expressed as mean ± SD. The maximum effect and the concentration that results in 50% of maximum effect were determined as described previously.1,2,7,18,20,24Comparison of two means was performed using the paired Student t  test. Comparison of several means was performed using one-way or two-way analysis of variance, when appropriate. Repeated-measures analysis of variance was used when required, and the post hoc  test used was the Newman–Keuls test. All P  values were two-tailed, and a P  value less than 0.05 was considered significant. Statistical analysis was performed using NCSS 2007 software (Statistical Solutions Ltd., Cork, Ireland).

We studied 49 young adult and 60 senescent rats. For assessment of mechanical variables with isolated left ventricular papillary muscles and protein expression with Western blot, we investigated 31 young adult and 42 senescent hearts. This difference was explained by the fact that it was more difficult to remove and obtain stable preparation in senescent rats. Nitric oxide assessment, staining, and imaging were performed in vivo  in 12 rats of each age. Nitric oxide assessment was performed in vitro  in 6 rats of each age.

Senescent rats had significantly higher body weight (551 ± 130 vs.  328 ± 35 g; P < 0.05) and heart weight (1,078 ± 185 vs.  664 ± 72 mg; P < 0.05) than young adult rats. Nevertheless, the heart weight–to–body weight ratio (2.1 ± 0.8 vs.  2.0 ± 0.2 mg/g; not significant [NS]) and left ventricular weight–to–body weight ratio (1.62 ± 0.52 vs.  1.63 ± 0.21 mg/g; NS) were not significantly different between young adult and senescent rats.

In vivo , the baseline echocardiographic characteristics were compared with young adult (n = 12) and senescent (n = 9) rats. The heart rate was not significantly different between young adult and senescent rats (340 ± 14 vs.  342 ± 11 beats/min, respectively; NS). Systolic function was preserved in senescent rats, as shown by the lack of a significant difference between young adult and senescent rats in left ventricular ejection fraction (86 ± 5 vs.  90 ± 5%; NS) and left ventricular fraction shortening (54 ± 5 vs.  56 ± 8%; NS). In contrast, diastolic function was altered in the senescent group, as shown by the significant prolongation of isovolumic relaxation time (22 ± 1 vs.  30 ± 4 ms; P < 0.05), the impairment in deceleration time of E (44 ± 7 vs.  32 ± 6 ms; P < 0.05), and the increased value of the E/A ratio (1.2 ± 0.1 vs.  2.7 ± 1.1; P < 0.05). Assessment of A, E/A ratio, and isovolumic relaxation time was technically impossible for two young adult rats. Left ventricular end-diastolic pressure was enhanced in comparison with the young adult rats, as shown by the increased E/Ea ratio (13.1 ± 2.5 vs.  17.7 ± 4.3; P < 0.05). In vitro , using left ventricular papillary muscle (n = 56 from young adult and n = 56 senescent hearts), the inotropic properties were significantly altered in the senescent group under both low-load conditions (maximum unloaded shortening velocity, 3.21 ± 0.39 vs.  2.60 ± 0.60 L^max/s; P < 0.05) and high-load conditions (maximum isometric active force normalized per cross-sectional area, 60 ± 17 vs.  51 ± 18 mN/mm²; P < 0.05) in comparison with the young adult group. The contraction time was increased in senescent rats as shown by the prolongation of both time to peak shortening (175 ± 16 vs.  206 ± 24 ms; P < 0.05) and time to peak force (150 ± 15 vs.  177 ± 25 ms; P < 0.05).

β-Adrenoceptor stimulation induced a marked positive inotropic effect in young adult rats in vivo  (table 1) and in vitro  (table 2and fig. 1A). This positive inotropic effect was markedly diminished both in vivo  (table 1) and in vitro  (table 2and fig. 1B) in senescent rats.

In vitro , l-NAME, l-VNIO, and 1400W per se  did not significantly modify the maximum isometric active force normalized per cross-sectional area in young adult or senescent groups (data not shown). With l-NAME, l-VNIO, or 1400W, the positive inotropic effect of β-adrenoceptor stimulation was not significantly modified in young adult rats (table 2and fig. 1A). In senescent rats, both l-NAME and l-VNIO partially restored the positive inotropic effect of β-adrenoceptor stimulation (table 2and fig. 1B). In contrast, this positive inotropic effect was not significantly modified by 1400W (table 2and fig. 1B).

BRL 37344 did not induce any significant inotropic effect in the presence of nadolol, a selective β¹- and β²-adrenoceptor antagonist, in young adult (101 ± 5%; NS) or senescent rats (98 ± 7%; NS). The positive inotropic effect of dibutyryl-cyclic adenosine monophosphate was not significantly different in young adult and senescent rats (fig. 2). BRL 37344 significantly decreased the positive inotropic effect of dibutyryl-cyclic adenosine monophosphate in senescent rats but not in young adult rats (fig. 2).

In agreement with the functional changes observed in the papillary muscle experiments, we found that protein expression of β¹-adrenoceptor was reduced by 33% in senescent hearts compared with young adult hearts (fig. 3). In contrast, β³-adrenoceptor protein expression was significantly increased in senescent hearts compared with young adult hearts (fig. 3).

In young adult hearts, the β-adrenoceptor stimulation did not induce any nitric oxide production in vivo  (fig. 4) or in vitro  (fig. 5A). In vitro , l-NAME abolished the nitric oxide production without any significant influence of l-VNIO or 1400W in comparison with the control group (fig. 5A). Nitric oxide production was significantly decreased by l-VNIO in comparison with the isoproterenol group (fig. 5A).

In senescent hearts, however, nitric oxide production significantly increased with β-adrenoceptor stimulation, both in vivo  (fig. 4) and in vitro  (fig. 5B). In vitro , l-NAME abolished nitric oxide production. Both l-VNIO and 1400W decreased around 50% of global nitric oxide production (fig. 5B).

Both NOS1 and NOS2 protein immunoreactivities were significantly increased in senescent left ventricular myocardium in comparison with young adult rats (fig. 6).

In the current study, we confirmed that the positive inotropic effect of β-adrenoceptor stimulation was altered in vivo  and in vitro  in senescent rat heart. Both in vivo  and in vitro , we provide evidence for involvement of the β³-adrenoceptor in the decreased positive inotropic effect of β-adrenoceptor stimulation in senescent heart, in parallel to down-regulation of β¹-adrenoceptor and up-regulation of β³-adrenoceptor protein expressions. In vitro , NOS1 seems to be the functional isoform involved in the β³-adrenoceptor pathway. These findings suggest that the β³-adrenoceptor plays an important role in the β-adrenergic dysfunction associated with senescent heart.

In vivo , we have confirmed left ventricular diastolic dysfunction in senescent heart allowing a preserved left ventricular ejection fraction with both a “restrictive filling pattern” in pulsed-wave spectral Doppler mitral flow and increased left ventricular end-diastolic filling pressures.2,25,In vitro , we have confirmed inotropic abnormalities involving the prolongation of the contraction velocities related to lower Ca²⁺release from the sarcoplasmic reticulum,25,26slower cross-bridge cycling rate,26and decreased density of depolarizing potassium channels responsible for transient outward potassium current I^to.27,28The impairment in the maximum unloaded shortening velocity is attributable to a switch of the myosin heavy chain isoform with lower adenosine triphosphatase activity (β-MHC).26The decreased maximum isometric active force normalized per cross-sectional area results from the diminished myofibrillar content as a result of fibrosis and apoptosis.2,25The usual contrast between the normal systolic function in vivo  and the myocardial inotropic abnormalities in vitro  suggest the existence of neurohumoral compensatory mechanisms that may allow the heart to maintain a normal cardiac output.25 

In senescent heart, the positive inotropic effect of β-adrenoceptor stimulation is altered both in vivo  and in vitro .3,4,29,30In this context, dibutyryl-cyclic adenosine monophosphate induces comparable positive inotropic effects in young adult and senescent groups, suggesting that most of the abnormalities of the β-adrenergic pathway are located upstream of protein kinase A activation. While down-regulation of β¹- and β²-adrenoceptors has been well established,4we have shown for the first time that the β³-adrenoceptors are up-regulated in the left ventricular myocardium of senescent rats. The impairment in the inotropic effect induced by dibutyryl-cyclic adenosine monophosphate when BRL 37344 was used confirmed that the β³-adrenoceptor pathway is involved in senescent heart. The lack of a negative inotropic effect induced by BRL 37344 when a specific β¹- and β²-adrenoceptor antagonist (nadolol) was used could seem contradictory. In fact, nadolol inhibits the production of cyclic adenosine monophosphate induced by β¹- and β²-adrenoceptor stimulation. In this context, the concentration of cyclic adenosine monophosphate might be insufficient to induce any β³-adrenoceptor effect. On the other hand, when the cytosolic cyclic adenosine monophosphate concentration was restored using dibutyryl-cyclic adenosine monophosphate, the negative inotropic effect induced by BRL 37344 significantly decreased the positive inotropic effect induced by dibutyryl-cyclic adenosine monophosphate in senescent myocardium. As previously reported, nitric oxide production induced by the β-adrenoceptor stimulation is exclusively the fruit of the β³-adrenoceptor pathway.9Therefore, the increased nitric oxide production assessed both in vivo  and in vitro  with β-adrenoceptor stimulation supports the hypothesis that β³-adrenoceptor is involved in β-adrenergic dysfunction. Moreover, the dibutyryl-cyclic adenosine monophosphate concentrations used in our study are thought to produce a maximal positive inotropic effect in adult rats.7,20Nevertheless, cytosolic cyclic adenosine monophosphate concentration in senescent myocardium is impaired because of both the decreased β¹-adrenoceptor–Gs protein coupling and altered activity of adenylyl cyclase.3,15,16Therefore, the magnitude of the negative inotropic effect of β³-adrenoceptor stimulation could be potentially underestimated. In contrast and as demonstrated previously, the contribution of β³-adrenoceptor in young adult rat myocardium is insignificant.7 

In our recent study of ischemic heart failure in the senescent rat, we demonstrated that NOS1, coupled to sarcoplasmic caveolin-3, was up-regulated in comparison with the matched control group, whereas NOS3 expression was decreased.11In another study performed in diabetic cardiomyopathy, we reported that nitric oxide production induced by β³-adrenoceptor stimulation was exclusively issued by NOS1 coupled to sarcoplasmic caveolin-3.7In this study, using electron paramagnetic resonance in vivo  and in vitro  11further provided evidence that nitric oxide production induced by β-adrenoceptor stimulation involves NOS1 and/or NOS2 in senescent rats. However, we could not be more specific in our conclusions because only around 50% of the nitric oxide production recorded was induced by β³-adrenoceptor stimulation. The rest of nitric oxide was from other sources blocked by an unspecific NOS inhibitor (l-NAME). Using immunolabeling and confocal microscopy, both NOS1 and NOS2 were up-regulated in senescent myocardium in comparison with young adult myocardium. Nevertheless, in vitro , using left ventricular papillary muscles, the contribution of NOS1 seemed exclusive to the β³-adrenoceptor pathway, whereas NOS2 inhibition did not significantly increase the positive inotropic effect of the β-adrenoceptor stimulation. Therefore, NOS2 contributed to the nitric oxide production but independently of the β³-adrenoceptor pathway. Our findings support the idea that cardiac NOS1-derived nitric oxide is involved in the autocrine regulation of β-adrenergic (β¹- and β³-adrenoceptor subtype) contractile responses in senescent heart and may explain, in part, the increased hemodynamic instability associated with the higher quantity of catecholamine during the perioperative period or in critical care during septic shock.31–33Therefore, these findings suggest that part of the marked altered response to the β-adrenergic stimulation could be corrected by the antagonism of the β³-adrenoceptor pathway and could at least partly restore cardiac output by the inotropic effect induced. Further studies are needed to confirm these hypotheses in humans.

The following points should be considered when assessing the clinical relevance of our results. First, this study was performed in rat myocardium, which differs from human myocardium. Second, a part of this study conducted in vitro  only dealt with intrinsic myocardial contractility. The inotropic effects observed on cardiac function using different adrenoceptor agonists or antagonists and different NOS inhibitors were independent of several in vivo  factors, such as variations in cardiac loading, the autonomic nervous system, and compensatory mechanisms.25Nevertheless, the confluent results obtained using five different technologies are noteworthy. Third, despite the fact that the magnitudes of adenylate cyclase activity, peak calcium transients, and systolic cell shortening induced by dobutamine, a partial agonist of β-adrenoceptors, are known to be slightly less important than with isoproterenol, a full agonist of β-adrenoceptors,34,35we made the choice to use these two different nonselective β-adrenoceptor agonists in vivo  and in vitro , respectively, because dobutamine is classically used in vivo  for β-adrenoceptor stimulation in stress echocardiography7,10,18and isoproterenol is commonly used in vitro  for β-adrenoceptor stimulation.7,18,20,36Anyway, the positive inotropic effect was significantly decreased in vivo  with dobutamine as well as in vitro  with isoproterenol. Fourth, part of the study was performed during halogenated anesthetic agent exposure, which is liable to interfere with the β-adrenergic stimulation in different kinds of cardiomyopathy.20,37However, our data obtained in vivo  using a halogenated anesthetic agent is in agreement with those obtained in vitro . Fourth, inhibition of β³-adrenoceptor could decrease its protective effect against arrhythmia in senescent heart as reported in acute myocardial infarction.35Further studies are needed to test this hypothesis.

In conclusion, in senescent heart, β³-adrenoceptor plays an important role in the altered contractile response of β-adrenergic stimulation via  induction of NOS1-nitric oxide.

The authors thank David Baker, D.M., F.R.C.A. (Staff Anesthesiologist, Department of Anesthesiology and Critical Care, Centre Hospitalier Universitaire Necker-Enfants Malades, Paris, France), for reviewing the manuscript. The authors also thank Sophie Besse, Ph.D. (Associate Professor, Laboratoire de Recherche sur la Croissance Cellulaire, la Réparation et la Régénération Tissulaires, Unité Médicale de Recherche Centre National de Recherche Scientifique 7149, Université Paris 12–Val de Marne, Créteil, France), for donating some of the senescent rats.