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Cardiovascular Research 1999 42(1):121-129; doi:10.1016/S0008-6363(98)00291-0
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Modulation of the pacemaker current If by β-adrenoceptor subtypes in ventricular myocytes isolated from hypertensive and normotensive rats

Elisabetta Cerbaia, Roberto Pinoa, Maria L Rodriguezb and Alessandro Mugellia,*

aDepartment of Preclinical and Clinical Pharmacology, University of Firenze, Firenze, Italy
bInstitute of Pharmacology and Toxicology, Universitad Complutense de Madrid, Madrid, Spain

* Corresponding author. Tel.: +39-055-4271-264; fax: +39-055-4271-285; e-mail: mugelli@server1.pharm.unifi.it

Received 9 July 1998; accepted 7 September 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Both β1- and β2-adrenoceptors (β1-AR and β2-AR) are functionally present in human and rat ventricular myocytes. The two receptor subtypes are differently regulated during the development of myocardial hypertrophy and failure. If is expressed in human and rat ventricular myocytes. In hypertrophied myocytes isolated from old spontaneously hypertensive rats (SHR) the density is much larger than in age-matched normotensive Wistar Kyoto (WKY). Due to the possible relevance of If as an arrhythmogenic mechanism in the rat and human ventricle, we studied and compared the effects of β1-AR and β2-AR stimulation on If in both hypertrophied and normal left ventricular myocytes of 18-month old SHR and WKY. Methods: The whole-cell configuration of the patch-clamp technique was employed. Noradrenaline (NA, 1 µM) was used to stimulate β1-AR and isoprenaline (ISO, 1 µM) in the presence of the β1-AR antagonist CGP 20712A (0.1 µM) to stimulate β2-AR. Results: In SHR, NA increased If by causing a 10.8±0.9 mV (n=10) positive shift in the voltage of maximal activation (V1/2); this effect was completely reversed by CGP 20712A. β2-AR stimulation was effective in seven out of 13 cells tested, where it caused a small positive shift in V1/2 (4.0±1.7 mV). Cyclopentyladenosine (CPA), a selective A1-receptor agonist, reversed the effect of NA; the antiadrenergic action of CPA was abolished in cells pre-incubated with pertussis toxin (PTX) to block inhibitory G proteins (Gi). In PTX-treated cells the shift in V1/2 caused by both β2-AR (9.6±1.7 mV, n=6, p<0.05) and β1-AR (17.6±1.9 mV, n=7, p<0.05) was significantly greater than in control cells. Both β-AR subtypes modulated If activation also in WKY: β1-AR shifted V1/2 by 16.0±1.4 mV (n=15) and β2-AR by 4.2±1.1 mV (n=7). However, in PTX-treated WKY cells only the β2-AR effect was potentiated (shift in V1/2: 11.4±1.4 mV, n=9, p<0.01), while the β1-AR response was unchanged (18.9±4.2 mV, n=5, n.s.). Conclusions: If expressed in SHR hypertrophied ventricular myocytes is modulated by catecholamines mainly through the stimulation of the β1-AR subtype. The β1-AR response is, however, significantly lower than that observed in myocytes from normotensive rats, probably as a consequence of the presence of an increased inhibitory activity of Gi proteins. This post-receptorial control may be seen as a mechanism to limit the arrhythmogenicity of β-AR stimulation in myocardial hypertrophy and failure.

KEYWORDS β-AR, β-adrenoceptor; β1-AR, β1-adrenoceptor; β2-AR, β2-adrenoceptor; CPA, Cyclopentyladenosine; Gi, Inhibitory GTP-binding protein; Gs, Stimulatory GTP-binding protein; gf,max, Maximal specific conductance of If; ISO, Isoprenaline; NA, Noradrenaline; PTX, Pertussis toxin; SHR, Spontaneously hypertensive rats; V1/2, Voltage of half maximal activation of If; WKY, Wistar Kyoto rats


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
The development of myocardial hypertrophy in response to chronic pressure overload is a complex adaptive phenomenon and a predictor of the progression to cardiac failure [1]. Most of the alterations observed in the hypertrophied myocardium anticipate those found in the failing heart.

A diminished response to β-adrenergic stimulation has been consistently found in the human failing heart [2–4]. This phenomenon has been attributed to the development of several signal transduction defects in the β-AR related pathway, such as the down-regulation of β-AR and the up-regulation of the two important regulatory systems, the type 1 β-AR kinase (βARK1) and the inhibitory G protein (Gi) [2, 3, 5–10]. The down-regulation of β-AR density is specifically limited to the β1-AR subtype, the density of β2-AR subtype remaining unchanged [2, 11]. Even if data are not available in humans, evidence that the defects in β-AR pathway found in heart failure are already present in compensated hypertrophy due to pressure overload has been obtained in animal models, such as the spontaneously hypertensive rat [12, 13].

SHR is a suitable animal model for studying the progression from compensated hypertrophy to overt failure [14]. SHR develop left ventricular hypertrophy in response to pressure overload, through a process which evolves continuously during their life. At 18 months of age, SHR show a severe compensated left ventricular hypertrophy and, between the ages of 18 and 24 months, 57% of male SHR have evidence of cardiac failure. Data suggest that this model is also predictive of the alterations occurring in human cardiac hypertrophy and failure. In old SHR, the action potential duration is prolonged, due to a decrease in repolarizing transient outward current density [15]; a similar alteration has been observed in failing human myocytes [16, 17]. Moreover, left ventricular myocytes from old SHR with severe myocardial hypertrophy and/or failure show a marked diastolic depolarization phase due to an over-expression of the pacemaker channel, If [18, 19]. A similar current is present in left ventricular myocytes isolated from human explanted hearts [20, 21]. In left ventricular myocytes from SHR If is modulated by β-AR stimulation, which shifts its activation curve toward less negative potentials, thus increasing the contribution of If to the diastolic phase [18]. Indeed, old SHR show an increased sensitivity to the arrhythmogenic action of isoprenaline: the occurrence of spontaneous activity is concomitant with an increase in the steepness of diastolic depolarization [22]. Thus, the β-AR-mediated increase in If could represent an important arrhythmogenic mechanism in myocardial hypertrophy and failure.

Both β1- and β2-AR are present in human and rat ventricular myocytes and are involved in the modulation of the excitation–contraction coupling process. Recent studies have shown that β-AR subtype stimulation elicits distinct cellular responses in rat ventricular myocytes. While the contractile effect of β1-AR stimulation is mediated by the classic stimulatory G protein (Gs)-coupled cAMP-dependent signaling pathway [23, 24], β2-AR-stimulated increase in intracellular Ca2+ transient and contractility appears to be dissociated from the increase in cAMP and occurs in the absence of an increase in cAMP-dependent phospholamban phosphorylation [25]. Furthermore, β2-AR but not β1-AR, are simultaneously coupled to the PTX-sensitive Gi as well as to Gs. This pathway confers negative feedback to the positive inotropic effect of β2-AR stimulation [23, 24]. Whether these differences are responsible for the less arrhythmogenic effect of β2-AR stimulation compared to β1-AR is not known [26, 27].

Due to the possible relevance of the pacemaker current If as an arrhythmogenic mechanism in the rat and human ventricle [18, 20], we designed a study aimed to characterize the effects of both β1-AR and β2-AR stimulation on If in left ventricular myocytes of old SHR, and to compare them with the β-AR response in age-matched normotensive WKY. Such an approach could also provide a deeper understanding of: (1) the pharmacological modulation of the pacemaker current expressed in ventricular myocytes; (2) the alterations in the β-AR pathway induced by the development of myocardial hypertrophy and (3) the role of Gi in controlling the β2-AR mediated response.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Cell isolation
The investigation conforms to the rules for the care and use of laboratory animals of the European Community (86/609/CEE).

Single left ventricular myocytes were isolated from male Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats, of 18–19 months using a protocol based on previously described procedures [15, 28]. After killing the rat, the heart was rapidly excised, weighed, mounted in a Langendorff apparatus and perfused for 20 min with a low calcium solution (LCS, see Section 2.3) prewarmed to 37°C and equilibrated with 100% O2. The solution was then quickly changed to LCS plus 1 mg/ml collagenase (Type I, Sigma), 0.03 mg/ml dispase (Boehringer), 1 mg/ml albumin (fatty acid free fraction V, Serva) for 25–30 min. The left ventricle and the septum were removed with fine scissors, cut into chunks and the pieces were stirred in the LCS. Cardiomyocytes that appeared in the supernatant were purified by gravity sedimentation, collected and stored in LCS at room temperature. The isolated cells were either kept at room temperature in LCS, supplemented with 1 mM CaCl2 and 4% penicillin/streptomycin (Gibco BRL) and used within the day, or alternatively stored in a tissue culture incubator at 37°C in a cell culture medium (MEM, Gibco BRL, supplemented with 1 mM CaCl2 and 4% penicillin/streptomycin) for use in experiments 3–20 h after isolation. For pretreatment with pertussis toxin (PTX, Sigma-Aldrich), cells were incubated in the cell culture medium containing 0.5 µg/ml PTX at 37°C for at least 5 h.

2.2 Electrophysiological recordings
Cells were placed in an experimental bath on the platform of an inverted microscope (Nikon Diaphot TMD, Japan). The patch-clamp technique (whole cell recording) was used to measure electrophysiological properties of the isolated myocytes. Experiments were performed using a patch amplifier (Axopatch-1B, Axon Instruments, CA, U.S.A) interfaced to a 486 personal computer by means of a DAC/ADC interface (Labmaster Tekmar, Scientific Solutions). Data were viewed on-line on an analogic oscilloscope and on a computer screen. Experimental control, data acquisition and preliminary analysis were performed by means of the integrated software package pClamp (Axon Instruments, CA, USA). Cells were superfused with normal Tyrode’s solution or with a modified Tyrode’s solution during measurements of the hyperpolarization-activated inward current (If) (see Section 2.3). Temperature was maintained in the range of 36–37°C. Patch-clamp pipettes, prepared from glass capillary tubes (Garner Glass, CA, USA) by means of a two-stage vertical puller (Hans Otchoski, Homburg, Germany), had a resistance of 1.5–2.5 M{Omega} when filled with the internal solution (see Section 2.3).

For most of the experiments with drug application, the patch-clamped cell was superfused by means of a temperature-controlled micro-superfusor which allowed rapid changes to the solution bathing the cell; this enabled us to minimize current run-down and to reduce exposure of the other cells present in the experimental chamber to drugs.

(–)-Isoprenaline (ISO) and (–)-noradrenaline (NA) (Sigma–Aldrich) were dissolved in distilled water to get a stock solution with a final concentration of 10 mM. The stock solution, which contained ascorbic acid (1 mg/ml) as an antioxidant, was then diluted with Tyrode’s solution to get the final isoprenaline or noradrenaline concentration.

Cyclopentyladenosine (CPA) (RBI), a selective A1-adenosine receptor agonist [29], was dissolved in 50% ethanol to prepare a stock solution of 1 mM, which was then diluted with Tyrode’s solution to the final concentration. Stock solutions (10 mM) of the selective β1-AR and β2-AR antagonists CGP 20712A (kindly gifted by Ciba Geigy) [30]and ICI 118,551 (Tocris Cookson) [31]in distilled water were used to prepare the final Tyrode’s solutions used for some of the experiments.

2.3 Solutions
The composition of the solutions employed was as follows (in mM): Low Calcium Solution (LCS): NaCl 120, KCl 10, KH2PO41.2, MgCl2 1.2, D(+)-glucose 10, taurine 20, HEPES–NaOH, 10 (pH 7.0). Tyrode’s solution for hyperpolarization-activated inward current measurements: NaCl 140, KCl 25, CaCl2 1.5, MgCl2 1.2, BaCl2 5, MnCl2 2, 4-aminopyridine 0.5, glucose 10, HEPES–NaOH 5 (pH 7.35); this solution allowed for the reduction of interference from other currents, i.e., L-type calcium current, inward rectifier-like current and transient outward potassium current. Pipette solution: K-aspartate 130; Na2–ATP 5, MgCl2 2, CaCl2 5, EGTA 11, HEPES–KOH 10 (pH 7.2; pCa 7.0).

2.4 Data analysis and statistics
Data analysis and fitting were performed by using the program Origin 4.1 (MicroCal Software, MA, USA) running on a Pentium personal computer. For fitting functions, non-linear models of convergence to solutions were used.

To evaluate steady-state values of the hyperpolarization-activated current, data were fitted to an exponential decay. The difference between the extrapolated value and that of the current at the end of the hyperpolarization step was usually small (in the order of a few pA). Current amplitudes were measured as the difference between the value at the steady state and that at the beginning of the test pulse, and normalized with respect to the membrane capacitance value (see below). Specific conductance was determined as a function of membrane potential according to the following equation:

Formula
where gf is the conductance (in pS/pF) calculated at the membrane potential Vm, I the current density (in pA/pF) and Vrev the reversal potential of the fully activated current (–17.3 mV and –22.9 mV for SHR and WKY respectively) [18, 28].

A Boltzmann model based on the partition theorem according to the general equation:

Formula
was fitted to the activation data, where V (mV) is the test membrane potential, V1/2 (mV) is the fitted potential for half-maximal activation and k (mV) is related to the slope of the activation curve.

Cell membrane capacitance (Cm) was measured by applying a ±10 mV pulse starting from a holding potential of –70 mV. The current transient following this clamp protocol was fitted with a mono-exponential model to compute series resistance (Rs) and then Cm using the two equations given below:

Formula
and

Formula
where Ipeak is the maximum level of current (relative to the holding current) following the depolarization and {tau} is the time constant of the exponential current decay. The membrane capacitance values obtained have been used to compute ionic current densities (If density in pA/pF). Membrane capacitance, which is considered a suitable index of cell size and hypertrophy [18], was 298.2±12.8 pF in WKY (n=72) and 335.4±13.0 pF in SHR (n=69) (p<0.05).

All data are expressed as mean±standard error of the mean (SEM). Statistical analysis was performed by means of the Graph Pad Instat program, using Student’s t-test (grouped data) or ANOVA followed by Student–Neuman–Keuls multiple comparisons test. A probability value of less than 0.05 was considered significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Effect of selective β-AR subtype stimulation in SHR
The physiological β1-AR agonist NA was used to evaluate the effect of β1-AR stimulation on If in SHR myocytes. Preliminary experiments showed that alpha-adrenergic stimulation (10 µM NA or 30 µM phenylephrine in the presence of 1 µM propranolol) had no effect on If (data not shown); thus, all the experiments were performed in the absence of alpha-AR antagonists. If is a hyperpolarization activated current, and its amplitude increases progressively with increasingly negative steps. Fig. 1 (A) shows the current elicited during steps in the range of –60 to –90 mV, i.e., the range of diastolic potential of ventricular myocytes. In the presence of 1 µM NA (B), If amplitude is markedly increased. As expected, this effect was due to a shift in the activation voltage of If toward less negative potentials (18). This is shown in panel C, in which specific current conductance (gf, see Section 2) was reported as a function of the step potential. In ten cells, NA caused a rightward shift in the voltage of maximal activation (V1/2) of 10.8±0.9 mV, with no change in the maximal current conductance, gf,max (control: 35.3±7.7 pS/pF; NA: 35.4±8.3 pS/pF). The effect of NA was completely reversed by adding a selective β1-AR antagonist, CGP 20712A (0.1 µM) (30), to the superfusing medium, thus confirming that the NA-induced shift of If activation was due to the stimulation of the β1-AR subtype.


Figure 1
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  		Fig. 1 Effect of β1-AR stimulation on If recorded from a SHR ventricular myocyte. Panels A, B: current traces measured during hyperpolarization to the voltage indicated near each trace, from a holding potential of –40 mV, in control (panel A) and after superfusion with NA (panel B). Panel C: activation curve of If measured in control, in the presence of NA alone and NA plus CGP 20712A. Curves were obtained by plotting If specific conductance (in pS/pF) versus membrane potential (in mV). Here and in the next figures, lines represent fitting of experimental data points with a Boltzmann function (see Section 2).

 
To investigate the possible role of β2-AR stimulation on If modulation, the non-selective β-AR agonist ISO (1 µM), in the presence of 0.1 µM CGP 20712A, was used. Fig. 2 shows the results of such an approach. In panel A, currents were elicited by a double-pulse protocol, during which the cell was hyperpolarized first to –80 mV, to activate half of the current, and then to –120 mV, to activate If completely; the cell was continuously superfused with CGP 20712A and current traces were obtained before and after addition of 1 µM ISO. β2-AR stimulation increased the amount of current recorded during the step to –80 mV, and decreased the tail current measured at –120 mV: this behavior is typically attributable to a shift in the activation curve of If. However, this was not a consistent finding: β2-AR stimulation was effective in only seven out of 13 cells tested, in which it caused a relatively small positive shift in V1/2 (4.0±1.7 mV, see panel B) as compared to that caused by β1-AR stimulation.


Figure 2
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  		Fig. 2 Effect of selective β2-AR stimulation on If in SHR myocytes. Panel A: superimposed current traces recorded using the two-step protocol shown in the bottom, in a cell superfused with CGP 20712A alone and CGP plus ISO. Panel B: activation curves of If obtained from the same cell as in A.

 
3.2 Gi modulated the β-AR-mediated effect in SHR
Thus, in left ventricular myocytes from old SHR, NA caused a dose-dependent positive shift in V1/2 (Fig. 3). Stimulation of A1-adenosine receptors with CPA dose-dependently reversed the effect of NA: in the presence of 1 µM NA, the V1/2 shift was reduced to 5.4±2.4 mV (n=3) and 2.7±1.0 mV (n=3) by adding 0.2 µM and 1 µM CPA, respectively (Fig. 3). A typical example of the antiadrenergic effect of CPA is shown in Fig. 4. In panel A, the current elicited by a step to –80 mV was increased by NA; adding CPA (0.2 µM) partially reversed the effect of β-AR stimulation. Correspondingly, CPA was able to antagonize the positive shift in the If activation curve induced by 1 µM NA (panel B).


Figure 3
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  		Fig. 3 Summary of the effect of β1-AR stimulation and of the antiadrenergic effect of A1-receptor stimulation on If measured in SHR cells. Each column represents the mean (±SEM) shift observed in the presence of 0.1 or 1 µM NA, and with 1 µM NA plus 0.2 or 1 µM CPA.

 

Figure 4
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  		Fig. 4 Typical experiment showing the antiadrenergic effect of A1-purinergic stimulation on the β1-AR effect in SHR cells. Panel A: superimposed current traces showing If evoked by a two-step protocol (drawn in the bottom) in control, in the presence of NA alone and NA plus the selective A1-agonist CPA. Panel B: activation curves of If obtained from the same cell as in A.

 
These data suggest indeed that the activation of Gi proteins via the stimulation of A1-adenosine receptors modulates the β1-AR-mediated effect on If in SHR myocytes. As previously stated, β2-AR stimulation in rat ventricular myocytes has been shown to be coupled to both stimulatory and inhibitory G proteins [23]. To evaluate the role of Gi in the previously described response of β2-AR stimulation on If, myocytes were incubated with PTX. Fig. 5 shows the effect of selective β2-AR stimulation on If in PTX-pretreated cells. In panel A, typical recordings obtained in control, in the presence of CGP 20712A alone, of CGP plus 1 µM ISO and of CGP, ISO and ICI 118,551 are superimposed. In PTX-treated cells the effect of β2-AR stimulation was consistent and markedly greater than that observed in non-pretreated cells (see Fig. 2). The mean V1/2 shift was 9.6±1.7 mV (n=6), a figure significantly different from that obtained in control cells (4.0±1.7 mV, p<0.05). Adding 100 nM ICI 118,551, a selective β2-AR antagonist consistently blocked this effect: in four cells the mean V1/3 shift was reduced to 1.3±1.1 mV in the presence of the β2-AR antagonist. The PTX block of Gi, however, also potentiated the effect of β1-AR stimulation. As shown in Fig. 6, β1-AR stimulation with 1 µM NA in a PTX-pretreated cell caused a marked increase in If amplitude recorded at –80 mV. This effect was not antagonized by 1 µM CPA, as expected to occur in the presence of an irreversible Gi block. The shift in the activation curve (panel B) induced by NA was however significantly larger than that obtained in non-pretreated cells (see Fig. 1C) (17.6±1.9 mV, (n=7) vs. 10.8±0.9 mV, p<0.05).


Figure 5
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  		Fig. 5 Effect of inactivation of Gi by PTX-pretreatment of SHR myocytes on the β2-AR effect. Panel A: superimposed current traces showing If evoked by a two-step protocol (drawn in the bottom) in control, in the presence of CGP 20712A (0.1 µM), of CGP plus 1 µM ISO and CGP, ISO and ICI 118,551 (100 nM). Panel B: corresponding activation curves obtained from the same cell.

 

Figure 6
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  		Fig. 6 Effect of inactivation of Gi by PTX-pretreatment of SHR cells on the β1-AR effect. Panel A: superimposed current traces showing If evoked by a two-step protocol (drawn in the bottom) in control conditions, in the presence of NA alone and NA plus the selective A1-agonist CPA. Panel B: activation curves of If obtained from the same cell as in A.

 
3.3 The inhibitory effect of Gi is different in myocytes from hypertensive and normotensive rats
To obtain insight into the relevance of cardiac hypertrophy for the previously described results, experiments were repeated in left ventricular myocytes from age-matched normotensive WKY. As expected [18], If was expressed in more than 90% of the cells isolated from old WKY, but its density was significantly lower than in SHR: gf,max was 20.9±2.0 pS/pF in WKY (n=37) versus 31.0±3.6 pS/pF in SHR, (n=35, p<0.05), while V1/2 was similar in both groups (–87.9±1.4 mV in WKY vs. –86.1±1.2 mV in SHR, not significant). Fig. 7 compares the effects of 1 µM noradrenaline on If in WKY cells, non-pretreated (left panels) or pretreated (right panels) with PTX. Top panels show current elicited by the typical two-step protocols, and the bottom panels the corresponding activation curves. It is evident that NA causes a similar increase in If recorded at –80 mV (panels A,C) and a similar positive shift in If activation curve in both control and PTX-treated cell. As in SHR, the effect of NA was completely blocked by adding 0.1 µM CGP 20712A (data not shown), thus suggesting that it was due to β1-AR stimulation. This was a consistent finding in all cells tested: the mean shift in V1/2 induced by NA was 16.0±1.4 mV in control cells (n=15) and 18.9±4.2 mV in PTX-treated cells (n=5) (not significant).


Figure 7
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  		Fig. 7 Typical experiments showing the effect of β1-AR stimulation on If recorded from WKY ventricular myocytes. Left panels: current traces (A) and corresponding activation curves (B) measured in a control cell, in the absence (m) and presence (l) of NA. In A, the holding potential was –40 mV and the two voltage steps to –90 mV and –130 mV. Right panels: current traces (C) and corresponding activation curves (D) measured in a PTX-treated cell, in the absence (m) and presence (l) of NA. In C, the holding potential was –40 mV and the two voltage steps to –80 mV and –120 mV.

 
Fig. 8 summarizes the results obtained in WKY and SHR ventricular myocytes, incubated in control solution or in the presence of PTX. Panel A and B show, respectively, the effect of β1-AR and β2-AR stimulation on the voltage-dependence of If activation. It is evident that, in control cells, the effect of β1-AR stimulation (panel A) measured in SHR myocytes is significantly smaller than that observed in WKY. The irreversible Gi block by PTX is able to restore the response to β1-AR stimulation in SHR cells, which becomes quantitatively similar to that observed in WKY. Gi block by PTX in WKY does not modify the response to β1-AR stimulation.


Figure 8
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  		Fig. 8 Consequence of PTX-pretreatment of SHR and WKY myocytes on the β-AR mediated effect on If. Each column represents the mean (±SEM) shift in the voltage of half maximal activation (V1/2), with respect to the corresponding control value, induced by selective β1-AR (A) and β2-AR (B) stimulation. Open columns: non-pretreated WKY; hatched columns: PTX-treated WKY; grey columns: non-pretreated SHR; black columns: PTX-treated SHR. *p<0.05 vs non-pretreated cells of the same strain; {dagger} p<0.05 vs WKY non-pretreated cells.

 
However, Gi block by PTX caused a similar effect on If in both WKY and SHR myocytes when exposed to β2-AR stimulation (Fig. 8, panel B). The positive shift in V1/2 induced by ISO in the presence of CGP 20712 A was 4.2±1.1 mV in control WKY cells (n=7) and 11.4±1.4 mV in PTX-cells (n=9) (p<0.01). The potentiation of the β2-AR response caused by PTX in WKY (2.7 folds) was similar to that measured in SHR (2.4 folds).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
These results demonstrate that both β-AR subtypes modulate the pacemaker current which is expressed in hypertrophied ventricular myocytes from 18-month old SHR. Stimulation of β1-AR and β2-AR shifts the If activation curve toward less negative values, with no change in the amplitude of the maximal specific conductance (gf,max) of the current. Thus, the action of catecholamines on the pacemaker current in ventricular myocytes resembles qualitatively that reported in sino-atrial node myocytes [32]. The effect of the β1-AR subtype is, however, much greater than that of the β2-AR subtype. Previous data have already shown the dissimilarity between β1-AR and β2-AR-mediated effect in cardiac cells in which both subtypes are present. In the sheep heart, β1-AR (but not β2-AR) increased the slope of diastolic depolarization, the amplitude of delayed afterdepolarizations, induced spontaneous activity in Purkinje fibers [26], and accelerated the rate of relaxation of ventricular trabeculae [33]. In rat ventricular myocytes, the functional consequences of β1-AR and β2-AR stimulation on cellular calcium homeostasis are markedly diverse [27]. Some of these differences are explained by the simultaneous coupling of β2-AR to both stimulatory and inhibitory, PTX-sensitive G proteins; this latter pathway, which is not activated by the β1-AR subtype, might limit the effects mediated by the activation of the ‘primary’ pathway, involving the stimulatory G proteins and the cAMP cascade [23]. The present results further support this view: in fact, the effect of β2-AR stimulation is significantly increased in cells preincubated with pertussis toxin. However, the explanation for such PTX-mediated potentiation of the β2-AR effect on If is not obvious. Previous studies by Xiao et al. [23, 25], focusing on the β2-AR actions on Ca2+ current, Ca2+ transient and contraction in rat ventricular myocytes, excluded a direct effect of the β2-AR-coupled Gi on adenylyl cyclase. The authors postulate that this pathway could be responsible for the activation of a cAMP-independent mechanism, possibly involving a protein phosphatase. However, according to present knowledge [34–36], cAMP directly modulates the f-channel, without intervention of phosphorylating enzymes such as protein kinase A. Even more so, phosphorylation of the f-channel (obtained with a phosphatase inhibitor, calyculin A) has been shown to increase current conductance, but not to shift the activation curve on the voltage axis [34]. Those data indicated that "phosphatase inhibition increases If in a manner distinct from the direct cAMP pathway" [34], which shifts the If activation curve but does not affect its conductance. In our experimental condition, β-AR stimulation, either in control or in PTX-treated cells, caused a ‘cAMP-like’ effect, that is, a marked shift in the activation curve with no significant effect on gf,max. Thus it seems reasonable to speculate that the potentiation by PTX of the β2-AR mediated effect on If in ventricular myocytes is related to increased cAMP production.

In normal rats, the PTX-sensitive G protein does not seem to be activated by β1-AR [23], and consequently, the β1-AR mediated effect in PTX-pretreated cells is not changed. Even the aging-dependent decrease in the inotropic effect due to β1-AR stimulation does not appear to involve Gi proteins [37]. However, in the hypertrophied myocytes from old SHR the situation is quite different: our results clearly demonstrate that pretreatment with PTX does potentiate both β1-AR and β2-AR effects on If. This result may be not completely unexpected: in fact, an increase in the activity of inhibitory G proteins has been demonstrated to occur as a consequence of the development of myocardial hypertrophy and failure in rat and in human myocytes [6, 7, 38]. Thus, a tonically disease-activated PTX-sensitive G protein can attenuate the response to β-AR stimulation; its pharmacological blockade by PTX causes an increase in the response mediated either by the β1-AR or by the β2-AR subtype. To our knowledge, this is the first direct demonstration of such a phenomenon on β-AR modulation of membrane currents. Furthermore, our results exclude the involvement of a tonically activated PTX-sensitive G-protein in normotensive age-matched WKY, confirming the recent results of Xiao et al. [37]. In fact, pretreatment of WKY cells with PTX potentiates the β2-AR-mediated effect but not that caused by β1-AR. Thus, in normal 18-month-old rats the situation appears to be similar to that described for adult and aged rats [23, 37]. It may be supposed that aging and hypertension have distinct consequences as for increasing inhibitory regulatory systems of β-AR signaling. This hypothesis is supported by the demonstration that Gi are not up- regulated in SHR during the prehypertensive stage [38]and in healthy aged human and rat hearts [37, 39]. Since the response to β1-AR in SHR becomes similar to that measured in WKY only after PTX treatment (that is, after removal of the inhibitory activity of Gi) it seems that adenylyl cyclase desensitization by the alpha subunit of Gi is the major mechanism responsible for the attenuation of the β-AR response in severe cardiac hypertrophy. Obviously, other pathophysiological alterations, such as the down-regulation of β-AR [37, 38, 40]and a diminished Gs density or activity, may become operative in both old SHR and WKY. While the latter hypothesis is attractive and to be tested, present knowledge does not support a role for Gs in the phenomenon. For example, treatment of rats with catecholamines desensitizes adenylyl cyclase by a downregulation of β-AR, an increase in Gi{alpha} but no change in Gs{alpha} [41]. We have previously shown that the density of the pacemaker current If in ventricular myocytes isolated from old SHR hearts is linearly related to the severity of myocardial hypertrophy [18]. We hypothesised that If may represent an arrhythmogenic mechanism in myocardial hypertrophy and failure, particularly under the influence of β-AR stimulation [18, 22]. This hypothesis may hold true also for the human failing heart, where If is present [20]and over-expressed with respect to normal hearts [21, 42]. The present data add an important piece of information: the stimulation of the β1-AR subtype causes a significantly greater effect on If than the β2-AR subtype. Even after PTX treatment, the shift in the If activation curve observed with ISO plus CGP 20712A (β2) never reaches the amplitude of that caused by NA (β1). This observation is not trivial: in fact, in similar experimental conditions, PTX-induced potentiation of the β2-AR effect on Ca2+ current and contraction amplitude of rat myocytes is such that it becomes equal or even greater than that evoked by β1-AR stimulation [23]. Thus, it is tempting to speculate that the intrinsic activity of β2-AR stimulation on If is less effective than that of β1-AR stimulation, not only because the β2-AR subtype is simultaneously coupled to a PTX-sensitive Gi and to a Gs, but also because other post-receptorial mechanisms (e.g., cAMP compartmentation) are operative. Not withstanding the molecular mechanism(s) underlying these differences, an increase in the inhibitory tone due to Gi activity [6, 7](present results) and the relative up-regulation of β2-AR in myocardial hypertrophy and failure [9, 12, 43]may be finally seen as important adaptive changes counteracting the increased arrhythmogenic hazard present in this setting (i.e. the over-expression of the pacemaker current If).

Time for primary review 27 days.


   Acknowledgements
 
This study was supported by a grant from Telethon (Project no. 709); MLR was supported by a grant of the European Community (BIOMED Concerted Action Contract no. BMH4-CT96-0287). We wish to thank Dr. Barbara Otti for her assistance in some of the experiments.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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