The modulation of β-adrenoceptor signaling in the hearts of hindlimb unweighting (HU) simulated weightlessness rats has not been reported. In the present study, we adopted the rat tail suspension for 4 wk to simulate weightlessness; then the effects of simulated microgravity on β-adrenoceptor signaling were studied. Mean arterial blood pressure (ABP), left ventricular pressure (LVP), systolic function (+dP/dtmax), and diastolic function (−dP/dtmax) were monitored in the course of the in vivo experiment. Single rat ventricular myocyte was obtained by the enzymatic dissociation method. Hemodynamics, myocyte contraction, and cAMP production in response to β-adrenoceptor stimulation with isoproterenol or adenylyl cyclase stimulation with forskolin were measured, and Gs protein was also determined. Compared with the control group, no significant changes were found in heart weight, body weight and ABP, while LVP and ±dP/dtmax were significantly reduced. The ABP decrease, LVP increase, and ±dP/dtmax in response to isoproterenol administration were significantly attenuated in the HU group. The effects of isoproterenol on electrically induced single-cell contraction and cAMP production in myocytes of ventricles in the HU rats were significantly attenuated. The biologically active isoform, Gsα (45 kDa) in the heart, was unchanged. Both the increased electrically induced contraction and cAMP production in response to forskolin were also significantly attenuated in the simulated weightlessness rats. Above results indicated that impaired function of adenylyl cyclase causes β-adrenoceptor desensitization, which may be partly responsible for the depression of cardiac function.
- simulated weightlessness
- adenylyl cyclase
- cyclic adenosine 5′-monophosphate
increasing evidence demonstrates that microgravity leads to reduced cardiac contractility (7, 27, 28). The depressed contractility may be due to the changes of cardiac tissue, the low cardiovascular response to low circulating blood volume, and the impaired regulation of cardiac function. It is well known that β-adrenoceptor is a predominant receptor in regulation of cardiac function, and recent studies have suggested that the responsiveness of cardiac contractility to β-adrenoceptor stimulation is reduced after weightlessness (13, 26).
Once the cardiac contractility to β-adrenoceptor stimulation is reduced, β-adrenoceptor desensitization occurs. β-Adrenoceptor desensitization has been shown to be due to downregulation of the β-adrenoceptor itself or the impaired postreceptor events. A previous study has reported that density and affinity of β-adrenoceptor are not changed under the simulated microgravity (6), but the postreceptor events, such as Gs protein/adenylyl cyclase (AC)/cAMP cascades, which may also be responsible for β-adrenoceptor desensitization, are still not well understood.
Different extrinsic conditions may induce different changes in the β-adrenoceptor signaling pathway. There is evidence that showed that the function of the Gs protein, which mediates the action of β-adrenoceptor stimulation (10, 12, 14), is impaired in the hypertrophied heart (14). There has been no study on the changes of the Gsα-subunits, a biologically active Gsα-small of an apparent molecular weight of 45 kDa (23), in weightlessness. The role of AC, the enzyme that is activated by the Gs protein and in turn converts ATP into cAMP, is also not known in weightlessness. It has been documented that cardiac hypertrophy was associated with reductions in cellular cAMP (4) and impaired AC response to forskolin, an activator of AC (19). On the other hand, it has also been reported that no significant impairment of AC activity with forskolin stimulation presented in the right hypertrophied heart after chronic hypoxia (15, 22). On β-adrenoceptor stimulation, whether the Gs protein/AC/cAMP cascade in the weightlessness is changed or not warrants study.
In the present study, both in vivo and in vitro we delineated the postreceptor signaling mechanisms in the hearts of rats subjected to tail suspension for 4 wk, which has previously been shown to reduce cardiac contractility (28). With the use of the hemodynamics in vivo, and electrically stimulated twitch amplitude and cAMP in isolated ventricular myocytes in vitro as parameters, we determined the changes in ventricular myocytes of HU and control (CON) rats subjected to manipulations that activated β-adrenoceptor or AC. We also measured the Gsα isoform in the heart of CON and HU rats. Results from this study have provided evidence for the first time that the function of AC is impaired after hindlimb unweighting, which may be responsible for the β-adrenoceptor desensitization in the simulated weightlessness.
MATERIALS AND METHODS
Animal care and animal model.
Tail-suspended, hindlimb-unweighting rat model (15, 22, 5) was used to simulate microgravity in this study. Male Sprague-Dawley (SD) rats that weighed 150–180 g (from the animal center of the Fourth Military Medical University) at the start of the experiment were randomly divided into two groups. One group of the rats (n = 40) was subjected to tail suspension to simulate microgravity, while the control (n = 40) was maintained in room air. The technique of tail suspension (22) with modification from our collaborative laboratory has been described in detail previously (13, 20). The animals were maintained at about −30° head-down tilt with their hindlimbs unloaded. All animals received standard laboratory chow and water ad libitum and were caged individually in a room maintained at 23°C on a 12:12-h light-dark cycle. The hindlimb unloading period was 4 wk. All aspects of this study were reviewed and approved by Animal Care and Use Board of the Fourth Military Medical University.
Surgical procedure for in vivo experiments.
After SD rats were anesthetized with pentobarbital sodium (45 mg/kg ip), the surgical procedure was performed as previously described (17). The trachea was intubated and connected to a rodent ventilator (Jiangwan I ventilator; the Second Military Medical University, China) for artificial ventilation with room air (stroke volume, 10 ml/kg; 60 strokes/min). The temperature of the heating pad was adjusted to 37°C by a temperature controller. Arterial blood pressure was continuously monitored via a saline-filled catheter (PE-50) inserted into the right femoral artery, which was connected to a pressure transducer (AB-621G, Nihon Kohden, Tokyo, Japan). ECG and heart rate were measured by standard limb lead II electrodes using an isolated ECG bioamplifier (V75–04; Coulbourn Instruments, Allentown, PA). PE-50 catheters (Becton Dickinson, Franklin Lakes, NJ) were inserted into the right external jugular vein of each rat for drug administration and into the left ventricle (LV) from the left carotid artery for measurement of LV systolic pressure (LVP), systolic function (+dP/dtmax), and diastolic function (−dP/dtmax) with a pressure transducer (AB-621G, Nihon Kohden). All signals were sent to a recording system (RM6200, Nihon Kohden). Following stabilization for 15 min, the animal was injected with vehicle or isoproterenol (15 μg/kg iv) through the left external jugular vein. All of the following determinations were carried on by other authors, and the groups were blind.
Isolation of ventricular myocytes.
Ventricular myocytes were isolated from the hearts of male SD rats using a collagenase perfusion method described previously (8). Immediately after decapitation, the hearts were rapidly removed from the rat and perfused in a retrograde manner at a constant flow rate (10 ml/min) with an oxygenated Joklik modified Eagle's medium supplemented with 1.25 mM CaCl2 and 10 mM HEPES at pH 7.2 at 37°C for 5 min. This was followed by 5-min perfusion with the same medium free of Ca2+. Collagenase was then added to the medium to a concentration of 125 U/ml with 0.1% (wt/vol) bovine serum albumin (BSA). After 35–45 min of perfusion with the medium containing collagenase, the atria were discarded. The ventricle tissues were dissociated by shaking in an oxygenated collagenase-free solution for 5 min at 37°C. Ventricular tissues were cut into small pieces with a pair of scissors followed by stirring with a glass rod for 5 min. The procedure separated the ventricular myocytes from each other. The residue was filtered through 250-mm mesh screens, sedimented by centrifugation at 100 g for 1 min, and resuspended in fresh Joklik solution with 1% BSA. More than 70% of the cells were rod shaped and impermeable to trypan blue. The Ca2+ concentration of the Joklik solution was increased gradually to 1.25 mM within 40 min.
Measurement of twitch amplitude of contraction.
After isolation of myocytes for 2 h, measurement of myocyte twitch amplitude was performed with the use of an optical video system, as described previously (21, 24). The myocyte was placed in a perfusion chamber at room temperature under an inverted microscope (Nikon) and perfused at a rate of 2 ml/min with a Krebs bicarbonate buffer that contained (in mM) 117 NaCl, 5 KCl, 1.2 MgSO4, 1.2 KH2PO4, 1.25 CaCl2, 25 NaHCO3, and 11 glucose, with 1% dialyzed BSA and a gas phase of 95% O2-5% CO2, pH 7.4. The myocyte was field electrically stimulated at a rate of 0.2 Hz with platinum electrodes connected to a voltage stimulator. The twitch amplitude was measured with an automatic video analyzer (25). As a prerequisite for the proper operation of this automatic analyzer, the image of cardiac myocytes projected on the video camera (SSCM370CE; Sony) and observed on the video monitor (PVM-145E; Sony) was first rotated to align horizontally and paralleled to each of the video raster lines. This was achieved by interposing a k-mirror (or a dove prism) between the microscope eyepiece and the video camera. The video analyzer was interposed between the video camera and the video monitor, and it generated a positionable rectangular window that was observed on the video monitor together with the image of the cell. Light-dark contrast at the edge of the myocyte provided a marker for measurement of the amplitude of motion. The amplitude of the marker was directly proportional to the dark image of contraction, and the action was in real time. The traces of twitch amplitude were recorded with the use of a two-channel amplifier recorder system. The amplitude of myocyte motion remained unchanged for at least 10 min, indicating the stability of the preparation.
Assay of cAMP.
The measurement of cAMP was performed according to the method described previously (2, 21, 25). Samples that contained 3 × 106 to 6 × 106 freshly isolated ventricular myocytes after 1.25 mM Ca2+ loading were incubated in an atmosphere of 5% CO2-95% air for 2 h. Isoproterenol (0.1–10 mM) or forskolin (1–100 mM) was added and incubated for 10 min. At the end of the treatment, the cells were centrifuged for 5 s at 100 g. The medium was aspirated, the sediment was resuspended in ice-cold Krebs solution, and the cells were centrifuged again for 5 s at 100 g. The supernatant was aspirated. Ice-cold ethanol-HCl (0.5 ml) was added, mixed, and left to stand for 5 min at room temperature. The supernatant was centrifuged at 3,000 g for 5 min and collected with a pipette. The precipitate was washed with 0.5 ml of ethanol-water (2:1), mixed, and centrifuged at 3,000 g for 5 min. The supernatant was also collected and added together. Finally, it was evaporated to dryness at 55°C under a stream of nitrogen. The sediment was stored at −20°C for assay of cAMP. The pellets were neutralized in 0.1 N NaOH for protein determination by the method of Lowry et al. (18), using BSA as a standard. For determination of cAMP, a competitive binding assay with a kit from Amersham was used. Briefly, 50 ml of 0.5 M Tris (4 mM EDTA) was added to 50 ml of each sample on ice, followed by 50 ml of [3H]cAMP and 100 ml of binding protein. The samples were vortexed for 5 s, placed in an ice bath, and allowed to incubate for 2 h. Charcoal suspension of 100 ml was added. The samples were vortexed for 10 s again and centrifuged at 12,000 g for 2 min at 4°C. Samples of 200 ml supernatant were removed for scintillation counting.
In the present study, we measured the levels of Gs protein in washed particulate membrane (WPM), which was prepared according to the method of Kumar et al. (16), and the β-actin was used as reference protein. To prepare WPM from isolated ventricular myocytes, the freshly isolated myocytes were centrifuged at 100 g, and the pellet was washed twice with a sucrose-Tris medium (0.25 M sucrose and 25 mM Tris·HCl, pH 7.5). The washed cell pellet was suspended and homogenized in a hypotonic membrane buffer (that contained 20 mM Tris·HCl, pH 7.5, 5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, and protease inhibitor: 0.4 mM phenylmethylsulfonyl fluoride) with an Ultra Turrax homogenizer in three bursts (5 s each) at intervals of 30 s. The homogenate was then centrifuged at 100 g for 5 min to separate the unbroken myocytes, and the supernatant was centrifuged again at 40,000 g for 25 min. The pellet was then washed once with the membrane buffer (50 mM Tris·HCl, and 150 mM NaCl, pH 7.4), recentrifuged, and finally suspended in the membrane buffer before storage at −70°C for immunoblotting of specific Gs protein isoform. Protein was determined by the method of Bradford (3) before using these fractions. For immunoblotting of Gs protein, SDS gel electrophoresis of polypeptides was performed on 12% polyacrylamide gels prepared according to the method of Pei (21). Twenty to fifty microliters of samples, each containing 100 mg protein, was added with the same amount of the sample loading buffer (the reducing buffer), which was heated for 5 min at 95°C. The solution was added into a single lane of the gel. Electrophoresis was performed, after which polypeptides were transferred onto a nitrocellulose membrane. The membrane was then washed with a Tris buffer solution (TBS) and incubated with TBS that contained 5% nonfat dry milk (blocking buffer), a procedure that blocks the nonspecific protein binding sites on nitrocellulose. A Gs protein antibody that recognizes predominantly small isoforms of Gsα antibody (from Calbiochem) was used for immunoblotting. The Gs antibodies at 1:1,000 dilution in the blocking buffer were incubated with the blot for 4 h at room temperature. After a wash with TBS that contained 0.5% Tween 20 and after several rinses in TBS, the nitrocellulose was incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG diluted at 1:5,000 in the blocking buffer for 2 h at room temperature. After several washes with TBS, the protein level on the nitrocellulose membrane was detected by a chemiluminescent substrate system. The nitrocellulose membrane was sealed with a working solution, which was prepared by mixing equal volumes of reagents A and B in the assay kit (Fuji, Hunt) in a plastic bag for further developing and fixing to the X-ray film (Kodak, Rochester, NY). Densitometric analysis was conducted on the protein bands for quantitative comparison.
Drugs and chemicals.
Type I collagenase, isoproterenol, propranolol, forskolin, phenylmethylsulfonyl fluoride, Tween 20, dithiothreitol, Tris, EDTA, and alkaline phosphatase-conjugated goat anti-rabbit IgG were purchased from Sigma Chemical. The [3H]cAMP assay system was purchased from Amersham International. Chemicals for SDS-PAGE, electrophoretic transfer of polypeptide, nonfat dry milk, and nitrocellulose membrane were purchased from Bio-Rad. Affinity-purified G protein antibody for Gsα-subunit was purchased from Calbiochem. All chemicals were dissolved in distilled water except forskolin, which was dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO was 0.1%, and at this concentration, DMSO had no effect on cAMP.
Values are expressed as means ± SD. In experiments concerning determination of twitch amplitude, one to three myocytes from a single rat were used. The values obtained from more than one myocyte were averaged, and the mean was used as a single entity for statistical analysis. Unpaired Student's t-test or ANOVA was employed to determine the differences among groups. Significance level was set at P < 0.05.
Body and heart weights in control and HU rats.
Body and heart weights are shown in Table 1. After 4 wk tail suspension, the body and heart weights were not significantly changed.
Effects of 4 wk HU on hemodynamics in anesthetized rats.
The hemodynamic parameters were continuously recorded. Table 2 summarizes mean arterial blood pressure (ABP), left ventricular pressure (LVP), systolic function (+dP/dtmax), and diastolic function (−dP/dtmax) in two groups. It was founded that ABP was unchanged, while LVP and ±LVdP/dtmax were all significantly decreased after 4 wk tail suspension (P < 0.01).
Effects of 4 wk HU on the cardiac inotropic response to isoproterenol in anesthetized rats.
In both control and HU animals, LVP, and ±LVdP/dtmax were all significantly increased following isoproterenol (15 μg/kg) intravenous administration (P < 0.01). However, these inotropic responses were significantly attenuated in the HU group (Table 2, Fig. 1, P < 0.01). In addition, the response of ABP following isoproterenol administration was blunted in the HU animals, and it is only significantly decreased in the control animals.
Effects of isoproterenol on the electrically induced contraction in single ventricular myocytes of the control and HU rats.
Electrical stimulation triggered contraction, and the contractile responses were significantly lower in the HU than in the control rats (Fig. 2A), consistent with the well-established notion that the contractility is reduced after suspension in vivo. Isoproterenol, a β-adrenoceptor agonist, at the range of 0.01–10 μM, dose dependently increased the electrically induced contraction in the isolated single ventricular myocyte (Fig. 2, A and B). This response was blocked by 1 μM propranolol, a β-adrenoceptor antagonist (data not shown). In ventricular myocytes isolated from the ventricle of the HU rats, the effects of isoproterenol on the contraction of the myocyte were significantly attenuated, indicating that the β-adrenoceptor desensitization occurred in the heart of the HU rats.
Gsα protein isoforms in ventricular myocytes of CON and HU rats.
Experiments on the contractile response in the present study suggested that β-adrenoceptor signaling cascades, namely, Gs protein/AC/cAMP, might be impaired in the heart of HU rats. To further determine whether or not the Gs protein and AC were impaired in the heart of HU rats, we first measured the content of Gsα-small (biologically active isoform) in the myocytes of HU rats. It was found that the density of the band of Gsα-small (45 kDa) in the membrane of ventricular myocytes was not significantly changed (Fig. 3, A and B).
Effects of forskolin on the electrically induced contraction in single ventricular myocytes of CON and HU rats.
AC is the enzyme that is activated by the Gs protein and in turn converts ATP into cAMP, in the myocytes of CON and HU rats. To further determine whether or not AC was impaired in the heart of the HU rats, we measured the contractile response to forskolin, a well-known activator of AC. It was shown that forskolin, at the range of 0.1–100 μM, dose dependently increased the electrically induced contraction in the isolated single ventricular myocytes (Fig. 4, A and B). In ventricular myocytes isolated from the ventricle of the HU rats, the effects of forskolin on the contraction of the myocyte were significantly attenuated, indicating that the AC or downstream signaling might be impaired in the heart of the HU rats.
Effects of isoproterenol and forskolin on cAMP accumulation in ventricular myocytes of CON and HU rats.
To further test whether AC was impaired in the heart of the HU rats, we determined the effect of forskolin on cAMP accumulation in the heart of both the control and the HU rats. We also determined the effect of isoproterenol. Isoproterenol (0.1–10 μM) and forskolin (1–100 μM) increased the cAMP accumulation in myocytes of both the control and the HU rats (Fig. 5, A and B). Like the contractile response, the responses to isoproterenol and forskolin were significantly blunted in the HU rats (Fig. 5, A and B).
The most interesting observations in the present study are 1) the stimulatory actions of isoproterenol, which stimulate the Gs protein, and of forskolin, which activates AC on the electrically induced contraction and cAMP production in the ventricular myocytes, are significantly attenuated; and 2) Gsα-small, the predominant biologically active Gsα isoform in the heart, is not changed in the HU rats. The observations indicate that, in the HU rats, the reduced responsiveness to β-adrenoceptor stimulation is due at least partly to an impaired function of AC. Once the cardiac contractility to β-adrenoceptor stimulation is reduced, β-adrenoceptor desensitization occurs. β-Adrenoceptor desensitization has been shown to be due to downregulation of the β-adrenoceptor itself or the impaired postreceptor events. A previous study has reported that density and affinity of β-adrenoceptor are not changed under the simulated microgravity (6). Together with the results in the present study, the reduced responsiveness to β-adrenoceptor stimulation is probably due to impaired postreceptor signaling mechanisms.
In our in vivo study, it was found that ABP was unchanged, while LVP and ±LVdP/dtmax were all significantly decreased after 4 wk hindlimb unweighting. It was more interesting to find that responses of ABP, LVP, and ±LVdP/dtmax to isoproterenol were significantly attenuated in the HU animals. Relatedly, in agreement with the recent studies in 2 wk HU mice (13) and 4 wk HU rats (26), experiments on the myocyte contractile responses to isoproterenol in vitro in the present study demonstrated that contractile responses to isoproterenol were impaired in myocytes isolated from HU rats. The results suggested that some downstream sites in β-adrenoceptor signaling cascades, namely Gs protein/AC/cAMP, might be impaired in the heart of HU rats.
To further determine whether or not the Gs protein and AC were impaired in the heart of HU rats, we first measured the content of Gsα-small (biologically active isoform) in the myocytes of HU rats. It was shown that the biologically active isoform, Gsα-small, was not changed after 4 wk hindlimb unweighting. This was in agreement with the previous finding that there was no change in Gsα-small after hypobaric hypoxia (14).
To further determine whether or not AC was impaired in the heart of the HU rats, we measured the contractile response to forskolin, which is well known to activate AC, the enzyme that is activated by the Gs protein and in turn converts ATP into cAMP, in the myocytes of the control and the HU rats. As expected, forskolin at 0.1–100 μM increased dose dependently the electrically induced contraction and cAMP in the ventricular myocyte of both control and HU rats. It is interesting to note that the cardiac responses to forskolin, at high concentrations (10–100 mM), were attenuated in the HU rats. The attenuated cardiac responses to high concentrations of forskolin in the HU rats suggested that only in a more active state is AC function really affected by hindlimb unloading.
In addition, an early explanation for the depressed contractility after simulated weightlessness is that it may be due to cardiac tissue atrophy (27), which is induced by 90-day (long term) hindlimb unweighting. It is notable that the heart weight and body weight were not significantly changed after 4 wk hindlimb unweighting in our present study. It is possible that the depressed contractility may not be due to atrophy, which did not exist after 4 wk hindlimb unweighting.
It should be stated that the results in our present study in 4 wk HU rats are different from the results in head-down bed rest humans in previous studies (1, 9). The data in these studies provided evidence that simulated microgravity for 5 or 14 days causes an increase in β-adrenoceptor responsiveness. The phenomenon of different results from different species is very interesting, and time-course study of the microgravity in HU rats (especially the microgravity for 5 or 14 days) is warranted.
The tail suspension model is a well-recognized model of microgravity for muscle and bone studies (20). However, in the heart, it is not certain that this is an appropriate model. At least two conditions may be added to microgravity. 1) Psychological stress may markedly increase the sympathetic tone and thus induce a β-adrenoceptor desensitization; and 2) an increased venous return in this model would add a volume overload to the heart that may also modify the adrenergic responsiveness. Further investigations are warranted to better clarify these paraphysiological processes.
1) The results clearly showed the decreased cardiac responses to isoproterenol and forskolin, strongly suggesting that there is an impaired adenylate function in the heart of the rat under microgravity, but unfortunately the β-adrenoceptor density and sensitivity were not measured. Although a previous study reported that density and affinity of β-adrenoceptor are not changed under simulated microgravity (6), the determination of density of β-adrenoceptor in the present study would be critical to support the conclusion. 2) Psychological stress and blood volume overload to the heart may be two important factors affecting the adrenergic responsiveness under microgravity, which should not be neglected. 3) A large part of the literature has reported similarly that microgravity results in a decrease in β-adrenergic responsiveness in animal models, which is different from the results reported in humans. The phenomenon of different results from different species is very interesting and should warrant attention from investigators.
The present study has provided evidence for the first time that, after hindlimb unweighting for 4 wk, which induces simulated weightlessness, the attenuated cardiac response to β-adrenoceptor stimulation is at least due to impaired function of AC. As for downstream of AC, such as calcium homeostasis in β-adrenoceptor signaling, further study is warranted.
This work was supported by grants (06Z042, 06MA226, 01MB129, and 06MA203) from Department of Health, General Department of Logistic, People's Liberation Army, and a grant from Shaanxi province (No. 2005KW-17) and grants (No. 30370580 and No. 30770802) from National Natural Science Foundation, People's Republic of China.
↵* W. Yin and J.-C. Liu contributed equally to this work.
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