© 1999 by European Society of Cardiology
Copyright © 1999, European Society of Cardiology
L-type calcium current and contractility in ventricular myocytes from mice overexpressing the cardiac β2-adrenoceptor1
aInstitut für Pharmakologie, Universitätsklinikum Essen, Essen, Germany
bInstitut für Pharmakologie und Toxikologie, Universitätsklinikum der TU Dresden, Dresden, Germany
cAbteilung für Nieren und Hochdruckkranke, Zentrum Innere Medizin, Universitätsklinikum Essen, Essen, Germany
dBabraham Institute, Cambridge, UK
eDepartment of Surgery, Duke University, Durham, NC, USA
fCardiac Medicine, National Heart and Lung Institute at Imperial College, Dovehouse Street, London, UK
* Corresponding author. Institut für Pharmakologie und Toxikologie, Universitätsklinikum der TU Dresden, Karl-Marx-Strasse 3, D-01109 Dresden, Germany. Tel.: +49-351-8832 830; fax: +49-351-8832 832. E-mail address: ravens@rcs.urz.tu-dresden.de (U. Ravens)
Received 18 March 1998; accepted 12 August 1998
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Abstract |
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 Top Abstract 1 Introduction2 Methods3 Results4 DiscussionReferences |
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Objectives: The reported increase in basal activity of hearts from transgenic mice (TG4) overexpressing the human β2-adrenoceptor (β2-AR) was explained by spontaneously active β2-ARs that stimulate the β-adrenergic cascade in the absence of an agonist. In order to examine altered myocardial function on a cellular level, we have investigated L-type calcium current (ICa,L) and cell shortening in ventricular myocytes from TG4 hearts. Myocytes from littermates (LM) and wild type animals (WT) served as controls. Methods: Cardiac β-AR density was measured by [125I]-iodocyanopindolol binding to ventricular membranes. ICa,L was assessed by standard whole-cell voltage clamp technique. Contractility was measured as cell shortening in ventricular myocytes and as force of contraction in electrically stimulated left atria. Results: Overexpression of β2-ARs was confirmed by an almost 400-fold increase in β-AR density. The β1:β2-AR ratio in WT mice was 71:29. Myocytes from TG4 and LM mice were similar in size as judged by membrane capacitance and two dimensional cell area. ICa,L amplitude was significantly lower in TG4 than in LM myocytes (with 2 mM [Ca2+]o –4.82±0.48 vs. –6.56±0.38 pA/pF, respectively). In TG4 myocytes, the ICa,L response to isoproterenol (1 µM) was almost abolished. Cell shortening was not different in physiological [Ca2+]o, but smaller in maximum [Ca2+]o when comparing TG4 to control myocytes. Basal force of contraction in left atria did not differ between TG4 and LM at any age investigated. In TG4 left atria the inotropic response to isoproterenol was also absent, whereas responses to high [Ca2+]o or dibutyryl-cAMP (1 mM) were present but reduced. The rate of spontaneous beating of right atria was elevated in TG4 mice. Conclusions: Since only spontaneous beating rate but neither basal ICa,L amplitude nor basal contractile activity were elevated, our data fail to reveal evidence for spontaneously active, stimulating β2-ARs in left atrium and ventricle. A contractile deficit unrelated to the β-adrenoceptor pathway is evident in TG4 myocytes and left atria.
KEYWORDS Transgenic mice; Myocardium; β2-Adrenoceptor overexpression; L-Type Ca2+ current; Myocyte shortening
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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In heart muscle, three subtypes of the β-adrenoceptor (β-AR), β1-AR, β2-AR and the putative β4-AR, activate adenylyl cyclase and increase the intracellular concentration of cyclic-adenosine monophosphate (cAMP) [1, 2]. Elevated concentrations of cAMP activate protein kinases to phosphorylate different effector proteins [3]. Phosphorylation of phospholamban disinhibits the Ca2+ ATPase of the sarcoplasmic reticulum (SR) leading to increased uptake of Ca2+ by the SR. Thus more Ca2+ is available for release during a contractile cycle resulting in a positive inotropic effect. In addition, activated protein kinase A phosphorylates L-type Ca2+ channels to increase Ca2+ influx during an action potential and this may also contribute to the positive inotropic effect.
Recently, Milano et al. [4]introduced transgenic mice (TG4) with 195-fold cardiac overexpression of the human β2-AR. The hearts of these animals were shown to contract more strongly than those of their littermates (LM) and could not be stimulated any further with isoproterenol [4–7]. However, the levels of increased basal activity differed substantially between studies. In one study, basal force of contraction in TG4 atria was similar to that of control atria maximally stimulated with isoproterenol [4], whereas a different group showed that basal left ventricular dP/dt of TG4 hearts was elevated only by some 10% of maximum isoproterenol response in control hearts [6]. The former authors explained their findings by suggesting that the enormous number of extra β2-ARs will increase the number of receptors in the active conformation, and hence the signal transduction cascade is stimulated even in the absence of an agonist [4].
In multicellular preparations, the presence of endogenously released transmitter cannot be excluded with all certainty, whereas isolated myocytes are definitely devoid of neurohumoral influences. Therefore, the aim of our study was to characterize the basal properties of L-type Ca2+ current (ICa,L) and cell shortening in myocytes isolated from transgenic (TG4), non-transgenic littermate (LM) and wild type (WT) mice. In TG4 myocytes basal ICa,L amplitude and contraction amplitude were not increased, but the responses to isoproterenol were blunted. These findings suggest, that adaptive changes of signal transduction may take place in hearts with overexpression of the human β2-AR. Some of the data have been reported in preliminary form [8, 9].
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2 Methods |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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All studies complied with the German and United Kingdom Home Office Regulations Governing the Care and Use of Laboratory Animals. TG4 and non-transgenic littermate mice had a mixed genetic background and their age ranged from 7 weeks to 20 months. TG4 mice were paired for breeding. The genetic status of the offsprings was checked prior to experimentation. For this purpose, mouse DNA was extracted from tail biopsies. TG4 mice were identified by Southern blot analysis with a probe to the SV-40 sequences (see [4]). The method did not allow us to discriminate between homo- and heterozygous genotype concerning the human β2-AR transgene. In some experiments, wild-type mice of the strain C57BL6 (aged 3–4 months) were employed as controls.
2.1 Radioligand binding assay
Heart muscle homogenate was prepared from the frozen ventricles of mouse hearts. Hearts were thawed in preparation buffer (5 mM Tris, 5 mM EDTA, pH 7.4), minced with scissors and homogenized with an Ultra-Turrax (1x10 s full speed, 2x20 s 2/3 speed). The homogenates were filtered through four layers of gauze and centrifuged at 10 000 g for 20 min. The pellets were then resuspended in incubation buffer (composition: NaCl 154 mM, Tris 10 mM, ascorbic acid 0.01%, pH 7.4), rehomogenized and diluted to 100 ml/g wet weight.
β-Adrenoceptor density was assessed in membrane preparations obtained from ventricular homogenate employing (–)-[125I]-iodocyanopindolol binding at six concentrations between 10 and 150 pM [10]. Specific activity of (–)-[125I]- iodocyanopindolol was 2200 Ci/mmol. Non-specific binding was measured with (±)-CGP 12177 (1 µM; 4-[3-t-butylamino-2-hydroxy-propoxy]benzimidazol-2-one). β-AR subtype distribution was determined by inhibition of (–)-[125I]- iodocyanopindolol binding (40–60 pM) by a wide range of narrowly spaced concentrations of the selective β2-adrenoceptor antagonist ICI 118551 (erythro-DL-1[7-methylindan-4yloxy]-3-isopropylamino-butan-2-ol) or the selective β1-adrenoceptor antagonist CGP 20712A (2-hydroxy-5[2-((2-hydroxy-3-(4-((1-methyl-4-trifluoromethyl)1H-imidazole-2-yl)-phenoxy)propyl)amino)ethoxy]-benzamide monomethane sulphonate). Binding assays were done in triplicate (see Michel et al. [10]). The protein content of the membranes was determined according to Bradford [11].
2.2 Cardiomyocytes
2.2.1 Isolation procedure
Ventricular myocytes were isolated by enzymatic dissociation either using a modification of the method described by Harding et al. [12]for myocyte contraction experiments or Wolska and Solaro [13]for electrophysiological measurements. With the latter method, mice received heparin (5000 units/kg body weight) by intraperitoneal injection 30 min before being killed by cervical dislocation. The heart was quickly excised, the aorta canulated, the heart mounted on a Langendorff apparatus and perfused with a nominally Ca2+-free, oxygenated preparation buffer (composition in mM: NaCl 133.5, KCl 4.0, NaH2PO4 1.2, MgSO4 1.2, Hepes 10.0, bovine serum albumin 1 mg/ml; pH 7.4) to remove all blood from the coronary circulation. After 5 min the perfusion solution was switched to preparation buffer containing collagenase (Worthington type I, 75 U/ml) and supplemented with 25 µM CaCl2. After 7 to 12 min of tissue digestion, the hearts were taken off the perfusion apparatus, all atrial tissue was removed and the ventricles were cut into small chunks that were gently agitated in preparation buffer supplemented with 100 µM Ca2+. Myocytes were harvested by pouring the suspension through a cheese cloth. In order to remove most of the collagenase and to increase [Ca2+]o gradually, the cells were washed two times. During this procedure the cells were permitted to settle under gravity for 10 min. After the supernatant was removed the cells were resuspended in preparation buffer containing 200 µM and 500 µM Ca2+, respectively, and were stored at room temperature until used. The remainder of the tissue was quickly frozen in liquid nitrogen and stored until required for the preparation of heart muscle homogenate used for radioligand binding assay.
2.2.2 Electrophysiological studies
Cardiomyocytes were transferred to a small plexiglas chamber mounted on the stage of an inverted microscope (IMT-2, Olympus). The bath was perfused continuously at a rate of 1.8 ml/min. The whole-cell patch-clamp technique [14]was used to measure membrane currents by means of a List EPC-7 amplifier (List Medical Instruments, Darmstadt, Germany) under the control of the PCLAMP 5.5 software (Axon Instruments, Foster City, USA). Patch electrodes were pulled from borosilicate filament glass and had tip resistances of 1.5–4.0 M
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At the beginning of each experiment, fast hyperpolarizing ramps (5 V/s, 5 ms) were applied in order to determine the membrane capacitance which was used to calculate current density. Series resistance compensation was set to 50–70%. The control current density was recorded 5 min after establishing the whole-cell configuration. The current density in the presence of isoproterenol was measured in the maximum of the effect (about 6 min after addition). In order to account for ‘run-down’ (for ICa,L see also Belles et al. [15]), time-matched control experiments without exposure to isoproterenol were performed. The run-down of ICa,L amounted to 10–15% in myocytes from littermates during the period of measurements. The current densities in the presence of isoproterenol (Fig. 1B) were not corrected for run-down.
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2.2.3 Measurement of calcium current ICa,L.
L-type calcium currents (ICa,L) were measured at room temperature (22±1°C) from a holding potential of –80 mV. For isolation of ICa,L from contaminating currents, INa and T-type ICa (if present) were inactivated by a 50-ms long prepulse to –40 mV, and K+ currents were reduced by replacing K+ with Cs+. The composition of the bath solution was (in mM): NaCl 137.0, CsCl 5.4, MgCl2 1.25, HEPES 10.0, glucose 10.0; the CaCl2 concentration was either 0.6 or 2.0 mM; the pH was adjusted to 7.4 with NaOH. The pipette solution had the following composition (in mM): CsCl 140.0, MgCl2 4.0, Hepes 10.0, EGTA 10.0, Na2ATP 4.0; the pH was adjusted to 7.3 with CsOH.
2.2.4 Measurement of cell shortening
Myocytes were allowed to settle at the bottom of a small Perspex chamber placed on the stage of an inverted microscope (Zeiss IM). The cells were allowed to attach themselves spontaneously to the floor of the chamber. After 5 min the chamber was perfused with oxygenated Krebs–Henseleit solution (composition in mM: NaCl 119, KCl 4.7, CaCl2 1, MgCl2 0.94, KH2PO4 1.2, NaHCO3 25, glucose 11.5, adjusted to pH 7.4 by equilibration with 5% CO2 and 95% O2). The temperature was maintained at 32.0±0.5°C by a heater placed just before the chamber inlet and by two thermocouples for feedback control of the heater. The myocytes were electrically stimulated 50% above their voltage threshold at 0.5 Hz by bipolar pulses through platinum electrodes placed alongside the bath. Unloaded cell shortening was measured with a video-camera/length detection system as described previously [16], but with spatial resolution 1 to 512 and time resolution 10 ms. Cells were allowed to stabilize for at least 5 min at 1 mM [Ca2+]o before they were challenged with increasing concentrations of [Ca2+]o to determine the maximum response in cell shortening. After returning to 1 mM [Ca2+]o, the maximum response to isoproterenol was determined.
2.3 Atria
Left and right atria were cut off and mounted in a 25-ml organ bath or in pairs into a 50-ml organ bath, one from a TG4 mouse, the other from a LM (dibutyryl-cyclic AMP experiments), with one end of the muscle clamped down between a pair of platinum electrodes and the free end tied to a strain gauge for force measurement. Left atria were stimulated at 2 Hz with a voltage 10% above stimulation threshold. Resting tension was adjusted to yield approximately half-maximum active force development. The stretching force required was not different in atria from TG4 or LM mice (i.e. 5 mN in both groups). The organ bath contained modified Tyrode solution of the following composition (in mM): NaCl 126.7, KCl 5.4, CaCl2 1.8, MgCl2 1.05, NaHCO3 22.0, NaH2PO4 0.42, glucose 5.0. The pH of the solution was maintained at 7.4 by passing a mixture of 5% CO2 and 95% O2 into the bath. Temperature was maintained at 37.0±0.2°C.
Right atria were mounted for measuring spontaneous beating frequency in the absence of any electrical stimulation by counting the number of contractions per minute.
2.4 Drugs
All drugs and chemicals were obtained from Sigma (Munich, Germany and Poole, Dorset, UK). (–)-[125I]-Iodocyanopindolol was purchased from New England Nuclear (Dreieich, Germany). ICI 118551, (±)-CGP 12177 and CGP 20712A were obtained from RBI (Natick, MA, USA).
2.5 Data analysis
Unless otherwise indicated, results are presented as mean values±S.E.M., where the numbers in parenthesis indicate the number of cells and the number of hearts. Data were combined for individual myocytes from a given preparation, so that statistical analysis was done using n values for hearts. Statistical differences were evaluated with Student’s t-test for paired or grouped data and considered significant if P<0.05. Whenever the necessary prerequisites for the conduction of a parametric test were not met, the corresponding non-parametric Mann–Wilcoxon–Whitney test was applied. Analysis of variance (ANOVA) followed by Tukey–Kramer post-hoc test was used for comparison between multiple groups.
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3 Results |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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3.1 Radioligand binding
Maximum binding of [125I]-iodocyanopindolol in mouse heart homogenate from control animals (WT and LM) aged 3–6 months amounted to 11.5±1.0 (n=5) without, and 10.3±1.0 fmol/mg protein (n=4) with collagenase treatment of the hearts prior to homogenization, the respective KD values were 29.8±2.7 and 32.4±5.0 pM. Since the differences between these values were not statistically significant, data from all control hearts were pooled resulting in mean values for maximum binding 11.0±0.7 fmol/mg protein and for KD 30.9±2.5 pM (n=9). Hearts from TG4 animals with an average age of 5 months had always been treated with collagenase prior to homogenization, and maximum binding amounted to 4519±336 fmol/mg protein, the KD value was 42.6±7.0 pM (n=4). Thus, the binding of [125I]-iodocyanopindolol was about 400 times higher in TG4 myocytes than in WT or LM controls. From displacement of [125I]-iodocyanopindolol binding with the subtype-selective antagonists, β1- and β2-ARs were defined as high affinity sites for CGP 20712A and ICI 118551, respectively, and were 70.5±3.0% and 29.2±3.2% of total β-ARs in control ventricles. The values estimated for TG4 hearts were 6±6% for β1-AR and 100±0% for β2-AR. The antagonist affinities for high and low affinity binding sites (pKi,high and pKi,low, respectively) are summarized in Table 1. These binding data clearly confirm the overexpression of β2-ARs in the hearts from transgenic mice.
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3.2 Calcium currents
Cardiac myocytes from the three groups of animals did not differ in size. This was estimated from two dimensional cell area [17, 18], i.e. 2552±1265 µm2 (mean±S.D.) for 141 cells from three LM hearts and 2611±1246 µm2 for 108 cells from three TG4 hearts, as well as from membrane capacitance where the respective values were 187.9±11.7 pF for WT (n=17 myocytes/8 hearts), 174.7±7.5 pF for LM (n=41 myocytes/12 hearts), and 186.8±13.4 pF for TG4 (n=34 myocytes/13 hearts). These findings suggest absence of hypertrophy in myocytes from TG4 animals. Fig. 1 shows the ICa,L amplitudes of individual myocytes measured at a test potential of +10 mV. In WT control mice average ICa,L amplitude was –3.54±0.60 (n=10 myocytes/4 hearts) at 0.6 mM Ca2+ and –6.82±0.41 pA/pF (n=7 myocytes/4 hearts) at 2 mM Ca2+ thus lying in the same range as reported by Nascimento [19]. At both Ca2+ concentrations tested ICa,L was significantly lower in myocytes from TG4 than from LM. Thus we could not detect evidence for enhanced basal activity of ICa,L in myocytes from mice overexpressing the β2-AR.
When the myocytes were exposed to a maximum effective concentration of isoproterenol (1 µM), ICa,L significantly increased in WT and LM controls but not in TG4 (Fig. 1B). The mean current increases in percent of predrug control measured at a constant test potential of +10 mV were 49.5±6.2% in WT; 48.8±5.3% in LM, and 10.5±6.8% in TG4 myocytes. Only in five individual myocytes of the latter group could a clear response be detected (increase of ICa,L>10%), whereas in the remaining 11 myocytes ICa,L remained constant or even declined after exposure to isoproterenol. These results demonstrate significant impairment of response to isoproterenol in myocytes from TG4 mice.
3.3 Myocyte shortening
One reason for the absence of ICa,L increase with isoproterenol in TG4 mice could have been that contractility was the only target for an overexpression-associated functional alteration. To verify this possibility we investigated contraction amplitude in myocytes from hearts of LM control and the transgenic group. The results are depicted in Fig. 2. Contraction amplitude (% cell shortening) did not differ between myocytes derived from LM and TG4 hearts when the cells were superfused with low Ca2+ solution (1 mM), but was significantly lower in TG4 (13.2±1.6%, n=17 cells/10 hearts) than in control myocytes (18.0±0.8%, n=21 cells/10 hearts) at maximally stimulating Ca2+ concentrations (4–8 mM in both groups).
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In TG4 myocytes responses to isoproterenol were blunted. Therefore, up to 1 µM of isoproterenol was used to ensure maximum stimulation of cell shortening, yielding an average contraction amplitude (in 1 mM [Ca2+]o) of 6.8±0.6% (42 cells/12 hearts). This was significantly lower than the maximum amplitude with isoproterenol (10–30 nM) in control cells (11.9±0.8%, n=28 cells/17 hearts, P<0.001) or with high Ca2+ in TG4 myocytes (13.2±1.6%, n=17 myocytes/10 hearts, P<0.01). The distribution of the responses is shown for individual myocytes in Fig. 3. Cell shortening increased in all myocytes from control and in many from TG4 hearts, however, the distribution of response amplitudes indicates that in TG4 myocytes, the responses were on average smaller and more often absent or negative than in control myocytes.
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At maximum Ca2+, there was no significant difference in the velocity of myocyte shortening or relengthening in myocytes from TG4 mice compared to LM controls. Velocity of shortening was 373±49 µm/s (16 cells/10 hearts) and 474±64 µm/s (19 cells/9 hearts) in TG4 and LM myocytes, respectively. The respective values for relaxation velocity were 293±48 µm/s (TG4) and 346±48 µm/s (LM). Fig. 4 shows an original twitch recording of a myocyte from a transgenic animal in which isoproterenol did not increase either amplitude or velocities. The increase in contraction with 4 mM Ca2+ was not sustained as often observed with strongly positive inotropic stimuli. Nevertheless, it shows that the cell was not contracting close to its maximum under conditions where it was unresponsive to isoproterenol. It should also be noted that the maximum contraction amplitude with Ca2+ is larger than average in this cell, and is within the range of the control values. This suggests that the loss of the response to isoproterenol and that to high Ca2+ can be independent.
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3.4 Atrial rate and force of contraction
To ensure that lack of elevated basal activity in myocytes from TG4 mice was not due to an artifact introduced by the isolation procedure, we also measured force of contraction in electrically stimulated left atria and spontaneous beating rate in right atria (Fig. 5). Basal force of contraction was similar in TG4 and LM left atria in animals from all age groups. In contrast, the spontaneous beating rate of right atria was larger in TG4 (544±36 beats/min) than in LM controls (425±17 beats/min, P<0.05; both groups 7 weeks of age), and this difference was not affected by age.
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In order to rule out that differences in stimulation frequency might be responsible for the discrepancy between our and previously published results [4, 5], we established force–frequency relationships in the absence and presence of 1 µM isoproterenol (Fig. 6). Mouse atria exhibited negative force–frequency relationships that were similar in TG4 and LM. In TG4 atria there was almost no inotropic response to isoproterenol whereas LM atria showed a marked increase in force of contraction especially at low frequencies of stimulation. It should be noted that TG4 atria, irrespective of exposure to isoproterenol never exhibited this characteristic feature of β-AR activation, suggesting the absence of spontaneous stimulation of the signaling cascade by β2-AR overexpression.
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) and LM controls (
). After these measurements the atria were allowed to recover at 2 Hz and were subsequently exposed to 1 µM (–)-isoproterenol (ISO) for at least 10 min before a second force–frequency relation was established. In TG4 atria there was almost no inotropic response to ISO (
) in contrast to LM atria, in which force of contraction was markedly enhanced especially at low frequencies of stimulation (
).So far we could not detect differences in basal contractility between TG4 and LM. In addition, TG4 atria did not respond well to isoproterenol. In order to test the maximum force development in atria we have employed inotropic stimuli downstream to β-AR activation. The cAMP analogue dibutyryl-cAMP (1 mM) produced a marked positive inotropic response in both control and TG4 atria (Fig. 7). This increase matched the effects caused by 7.8 mM [Ca2+]o and could not be enhanced any further by exposure to 7.8 mM [Ca2+]o in the presence of dibutyryl-cAMP (Fig. 7). Although the contraction amplitude increased in TG4 atria after exposure to dB-cAMP, the maximum response was significantly smaller than in LM (Fig. 8).
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4 Discussion |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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In the initial characterization of cardiac tissue from transgenic mice overexpressing the β2-AR, it was reported that right atrial heart rate was elevated and that the contractile response to isoproterenol was reduced or absent because the myocardium was in a maximally activated state in the absence of β-AR agonist stimulation [4]. This was attributed to an active state of the unoccupied β2-AR, which, with the greatly increased number of receptors, had reached a functionally significant level [5]. In the present study we confirm the increased right atrial frequency. In addition, we have also found a markedly reduced contraction response to isoproterenol in TG4, but this was not due to the myocardium working at maximum capacity because left atria still responded to dibutyryl-cAMP. In both isolated left atria and isolated ventricular myocytes, contraction at physiological Ca2+ levels was not significantly different between TG4 and LM. At maximally activating Ca2+, contractile force of the left atria, as well as contraction amplitude of ventricular myocytes, were actually lower in TG4 compared to control. In addition, at both extracellular Ca2+ concentrations, ICa,L amplitude of TG4 myocytes was significantly smaller than in myocytes from control animals. The response to dibutyryl-cAMP matched that of high [Ca2+]o (7.8 mM) demonstrating that the biochemical cascade downstream the adenylyl cyclase was intact in TG4 and that maximum responses are limited by the reduced Ca2+ response. It is not clear why TG4 myocytes exhibit lower maximum responses to high extracellular Ca2+ concentrations. Certainly, the reduced influx of Ca2+ via L-type Ca2+ channels represents a smaller trigger for Ca2+-induced Ca2+ release from the SR. The smaller ICa,L, demonstrated in this study, is in line with a markedly reduced single channel activity of L-type calcium channels observed in TG4 myocytes [20]. However, changes in Ca2+ sensitivity of the contractile proteins could also contribute.
In TG4 mice the phenotype of overexpressed β2-ARs was confirmed by radioligand binding assays. Total β-AR number was about 400-fold higher in TG4 than in control myocardium, compared to 195-fold in the original report [4], although absolute values for both control and TG4 were lower in the present study (11 vs. 180 fmol/mg protein control and 4519 vs. 52 800 fmol/mg protein TG4). Importantly, most binding studies were performed on collagenase-treated tissue at the end of the myocyte preparation, and there was no difference compared to binding data obtained in untreated tissue. This eliminates the possibility that enzymatic digestion had removed sarcolemmal β-ARs. In addition, we have performed an autoradiographic study on the cell preparation which indicates that β-ARs are markedly increased in all myocytes from TG4 ventricles (preliminary data, not shown). However, neither radioligand binding nor autoradiographic techniques exclude the possibility, that the receptors are located on an intracellular membrane, since both techniques used the lipophilic ligand iodocyanopindolol. Internalisation and sequestration of β-ARs are initial steps in the process of receptor desensitisation [21].
The β1:β2-AR ratio was estimated in ventricular homogenates and amounted to 71:29 in control mice, which does not necessarily indicate the actual β1: β2-AR ratio on cardiomyocytes, because at least in rat heart a large fraction of β2-ARs is known to be located on connective tissue and arterioles [22]. Nevertheless, the β1:β2-AR ratio is almost identical to the ratio described previously in mouse [23]and similar to the subtype ratio observed in homogenate from rat ventricles [22, 24]. The large scatter of the relative values of the β1:β2-AR ratio in TG4 (i.e. 6±6: 100±0) indicates that the density of β1-ARs cannot be measured precisely. Thus it is not clear whether the absolute number of β1-ARs is increased, decreased or unchanged.
Taken together our data suggest, that there had been adaptive changes in the working myocardium of TG4 animals to minimise the effects of the increased β2-AR number, but not in sinoatrial node. Is this change related to the β-AR pathway, or have there been alterations in excitation–contraction coupling which reduce contractility? The lower force maximum in the presence of dibutyryl-cAMP or high Ca2+ is more consistent with an impaired excitation–contraction machinery. The significantly lower ICa,L probably also contributes to the decrease in maximum contraction observed. However, the maximum contractile amplitude produced by isoproterenol was reduced to an even greater degree than that in high Ca2+, and in some cells was absent altogether. Similarly, ICa,L was unresponsive to β-AR stimulation. There has, therefore, been a specific loss of β-AR responsiveness in both atrial tissue and ventricular myocytes from TG4 animals. A similar finding was reported by Du et al. [6]who showed that LV dP/dt in TG4 mice, although slightly higher at baseline, did not respond to isoproterenol and was less than half the maximum induced by isoproterenol in controls. The blunted response to isoproterenol in TG4 mice from a similar baseline as in LM controls raises the question why β1-AR signaling, which mediates the main response in control cells, is suppressed in the transgenics. We could not exclude reduced β1-AR number, since the β1-AR density could not be measured accurately against the huge background of overexpressed β2-ARs. Desensitization, impaired coupling, downstream inhibition of the signal transduction cascade, or any combination of the above could also contribute. Preliminary experiments suggest that this may involve activated inhibitory guanine nucleotide binding proteins [25], which are upregulated in TG4 hearts [26].
At present we cannot give a definite explanation for the discrepancies between our finding of similar basal contraction amplitudes in TG4 and LM myocytes and atria, and the enhanced contractility of TG4 atria reported originally [4, 5]. Could the mice used for the present study differ from those used for the initial characterization by Milano et al. [4]? Our mice will be from a later generation than the original animals studied. Therefore, the genetic background of the mice employed in this study might have changed. Also, breeding over several generations may have allowed some selection to take place. However, litter sizes were similar between control and transgenic pairs, and there was no evidence of excess perinatal or adult mortality in our TG4 colony. Another possible explanation for the lack of enhanced contractile function reported here could be related to differences in gene dose, i. e. TG4 mice employed in earlier studies might have had a higher number of transgene copies in their genome. In addition, reviewing the previous literature [4–7], it remains unclear in most cases, whether experiments were carried out with homo- or heterozygotes, or — like in our study — with a putatively mixed population. However, we do not think that possible differences in gene dose can explain the discrepancies with earlier studies, since the phenotype, estimated as the level of overexpressed β2-ARs, was similar. Finally, although the mean age of our animals was somewhat higher than in the original report [4], we clearly demonstrated that atrial force of contraction was not elevated in TG4 mice even at the young age of 7 weeks (compare Fig. 5).
Our data revealed conflicting evidence for spontaneously active β2-ARs that lead to enhanced basal myocardial function: right atrial heart rate was elevated which is in accordance with the concept of spontaneously active β2-ARs, whereas ICa,L and basal force of contraction were not increased. In addition, the force frequency relationships of TG4 atria were devoid of the typical shape seen after β-AR stimulation in LM controls. These findings suggest that compensatory mechanisms might vary at different sites within the heart. It should be pointed out, however, that in mouse myocardium, β2-AR stimulation may not mediate any positive inotropic response at all as suggested previously from experiments with β1-AR knockout techniques [23]or receptor stimulation with subtype selective agonists [[25, 27], own unpublished data]. One possible explanation for this lack of positive inotropic response to β2-AR stimulation in mouse hearts could be that activation of the β2-AR subtype, which couples to both stimulatory and inhibitory G proteins [25, 27], causes opposing effects that compensate each other. Hence putative spontaneous activity of overexpressed β2-ARs does not necessarily produce any net functional change because of dual coupling to opposing signal transduction pathways. In addition, overexpression of β-ARs does not necessarily mimic all functional changes of receptor stimulation by agonists, as recently demonstrated by Mansier et al. [28]. With eightfold overexpression of the human β1-AR in murine atria, these authors found a two-fold higher basal contractility of left atria, but no differences in in vivo heart rate when compared to control mice. Therefore, lack of elevated basal ICa,L and contractility does not exclude spontaneously active β2-ARs. Spontaneous frequency of TG4 right atria was significantly elevated and there could be other significant changes in functions not investigated in the present study.
In conclusion, cardiac overexpression of the human β2-AR caused regional differences in functional modulation within the heart: beating rate of right atria was enhanced, but basal contractility of left atria and ventricular myocytes was not different from controls. Furthermore, the contractile responses to maximum stimulation with high Ca2+ concentrations or dB-cAMP, and ICa,L were depressed. Investigation of the processes by which this takes place, and particularly the mechanisms by which the overexpressed β2-ARs cause a decrease in function of the native β1-ARs, could give useful insights into β-AR cross-talk in vivo.
Time for primary review 30 days.
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This study was supported by IFORES grant 107.410.0 of the Medical Faculty of the University of Essen and by the Deutsche Forschungsgemeinschaft (Ra 222/8-1). AJK thanks the British Heart Foundation and SEH thanks the Welcome Trust for support. The authors gratefully acknowledge the excellent technical assistence of Sylvia Grunz, Romy Kempe, Barbara Langer, Iris Manthey, Peter O’Gara and Doris Petermeyer.
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1 See pages 3–5.
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