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Inotropic response to -adrenergic receptor stimulation and anti-adrenergic effect of ACh in endothelial NO synthase-deficient mouse hearts

Axel Gödecke , Thomas Heinicke , Andreij Kamkin †, Irina Kiseleva †, Ruth H. Strasser ‡, Ulrich K. M. Decking , Thomas Stumpe , Gerrit Isenberg † and Jürgen Schrader §

	ABSTRACT

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1. The functional consequences of a lack of endothelial nitric oxide synthase (eNOS) on left ventricular force development and the anti-adrenergic effect of acetylcholine (ACh) were investigated in isolated hearts and cardiomyocytes from wild type (WT) and eNOS knockout (eNOS-/-) mice.

2. eNOS expression in cardiac myocytes accounted for 20 % of total cardiac eNOS (Western blot analysis). These results were confirmed by RT-PCR analysis.

3. In the unstimulated perfused heart, the left ventricular pressure (LVP) and maximal rate of left ventricular force development (dP/dt_max) of eNOS-/- hearts were not significantly different from those of WT hearts (LVP: 97 ± 11 mmHg WT vs. 111 ± 11 mmHg eNOS-/-; dP/dt_max: 3700 ± 712 mmHg s^-1 WT vs. 4493 ± 320 mmHg s^-1 eNOS-/-).

4. The dobutamine (10-300 nM)-induced increase in LVP was enhanced in eNOS-/- hearts. In contrast, L-type Ca²⁺ currents (I_Ca,L) in isolated cardiomyocytes of WT and eNOS-/- hearts showed no differences after -adrenergic stimulation. Dibutyryl-cGMP (50 M) reduced basal I_Ca,L in WT cells to 72 ± 12 % while eNOS-/- I_Ca,L was insensitive to the drug. The pre-stimulated I_Ca,L (30 nM isoproterenol) was attenuated by dibutyryl-cGMP in WT and eNOS-/- cells to the same extent.

5. The Ca²⁺ (1.5-4.5 mM)-induced increase in inotropy was not different between the two experimental groups and -adrenergic receptor density was increased by 50 % in eNOS-/- hearts.

6. The contractile effects of dobutamine could be inhibited almost completely by ACh or adenosine. The extent of the anti-adrenergic effect of both compounds was identical in WT and eNOS-/- hearts. Measurement of I_Ca,L in isolated cardiac myocytes yielded similar results.

7. These data demonstrate that in the adult mouse (1) lack of eNOS is associated with increased cardiac contractile force in response to -adrenergic stimulation and with elevated -adrenergic receptor density, (2) the unaltered response of I_Ca,L in eNOS-/- cardiac myocytes to -adrenergic stimulation suggests that endothelium-derived NO is important in mediating the whole-organ effects and (3) eNOS is unimportant for the anti-adrenergic effect of ACh and adenosine.

	INTRODUCTION

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It is well established that NO released from the endothelium plays an important role in the regulation of vascular tone. This was most stringently demonstrated by the generation of mice deficient in endothelial NO synthase (eNOS), which develop hypertension (Huang et al. 1995; Shesely et al. 1996; Gödecke et al. 1998). Besides its role in the regulation of organ perfusion, NO might also modulate cardiac function (for review see Kelly et al. 1996). The cytokine-mediated depression of cardiac contractility has been attributed to elevated inducible NO synthase (iNOS) expression accompanied by a high local NO formation (Forfia et al. 1999). Similarly, studies on isolated rabbit papillary muscle (Ishibashi et al. 1993) and guinea-pig hearts (Kelm et al. 1997) revealed that high doses of NO resulted in a decrease of myocardial contractility.

In addition to the modulation of cardiac contractile force under pathophysiological conditions, an early report on isolated rat cardiac myocytes demonstrated that the spontaneous beating rate and the amplitude of contraction in response to -adrenergic receptor stimulation were attenuated by endogenously synthesized NO (Balligand et al. 1993). Studies in the canine or human heart in situ (Hare et al. 1995a, 1998; Keaney et al. 1996) demonstrated that NOS inhibitors increased dP/dt_max after intracoronary application of dobutamine or isoproterenol (isoprenaline), thereby supporting the observation of a catecholamine-antagonistic effect exerted by the L-arginine-NO pathway. However, Klabunde et al. found a negative inotropic effect of NOS (Klabunde et al. 1992) and guanylyl cyclase inhibition (Klabunde et al. 1998) in isolated isoproterenol-stimulated rat hearts. In a recent report, however, Vandecasteele et al. (1999), analysing the effect of isoprenaline on contractile force of papillary muscles of WT and eNOS-/- hearts, reported no alteration of basal or stimulated contractile force. Thus, the role of NO in the modulation of the catecholamine-induced increase in inotropy is still not unambigously solved.

In the heart, several sources of endogenous NO formation exist. The endothelial NO synthase (eNOS) constitutes the major NOS isoform expressed in the endocardial and coronary endothelium (Kelly et al. 1996) and, according to more recent results in rat and human (Seki et al. 1996; Wei et al. 1996), also in cardiac myocytes and in the specialized cells of the conducting system of the sinus and atrioventricular nodes. In addition, cardiac nerves have been shown to express the neuronal NOS (nNOS; Hassall et al. 1992; Ursell & Mayes, 1995). Thus, under physiological conditions, NO derived from different isoenzymes expressed in different cell types may modulate contractility in a paracrine and/or autocrine manner.

Besides modulation of the -adrenergic response, evidence has been provided that NO mediates the anti-adrenergic effect of ACh, also termed 'accentuated antagonism', since NOS inhibitors attenuated the inhibitory effect of the muscarinic agonist carbachol on inotropic -adrenergic stimulation (Balligand et al. 1993). This initial observation in isolated rat cardiac myocytes was verified in rabbit sinoatrial and atrioventricular nodal cells (Han et al. 1996), and in the canine heart in situ (Hare et al. 1995b). However, experiments performed in isolated rat cardiomyocytes to elucidate the role of NO in the anti-adrenergic effect refuted a contribution of NO (MacDonell et al. 1995). This controversy now extends to the analysis of I_Ca,L in cardiac myocytes isolated from eNOS-/- mice. While Han et al. (1998) reported a lack of muscarinic cholinergic regulation of I_Ca,L in cardiac myocytes isolated from eNOS-deficient mouse hearts, Vandecasteele et al. (1999), using the same strain of knockout mice (Huang et al. 1995), presented evidence of unaltered muscarinic attenuation of the isoprenaline-stimulated I_Ca,L in eNOS-deficient cardiac myocytes.

In view of the discrepant results in the literature we addressed the role of eNOS in -adrenergic stimulation and the anti-adrenergic effect of ACh in an independent strain of eNOS-/- mice generated in our laboratory (Gödecke et al. 1998). In contrast to earlier reports (Han et al. 1998; Vandecasteele et al. 1999) using isolated cardiac myocytes and papillary muscle preparations, we used the isolated perfused heart and assessed in this more intact model left ventricular contractile force, the response to -adrenergic stimulation, -adrenergic receptor density and the anti-adrenergic effect of ACh. In addition, experiments with isolated cardiac myocytes were performed to address the role of eNOS in adrenergic and muscarinic modulation of L-type calcium currents.

	METHODS

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Mice

The generation and functional characterization of eNOS-deficient mice has been described (Gödecke et al. 1998). eNOS-/- mice were backcrossed for seven generations into the C57BL6 background. C57BL6 mice served as WT controls. Mice weighing 24.8 ± 2.8 g (WT) and 25.2 ± 2.2 g (eNOS-/-) aged 3-6 months were analysed. The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996).

Expression analyses

For Western and RT-PCR analyses murine cardiomyocytes were prepared according to Piper et al. (1990). In brief, the aorta of urethane-anaesthetized (1.5 g kg^-1 I.P.) mice was cannulated in situ and hearts were perfused in a retrograde manner with a modified Ca²⁺-free Krebs-Henseleit bicarbonate buffer (mM: NaCl 110, KCl 2.6, KH₂PO₄ 1.2, Mg₂SO₄ 1.2, NaHCO₃ 25 and glucose 11). Subsequently, hearts were excised, perfusion was switched to a recirculation mode, and 0.1 % collagenase, 0.5 % bovine serum albumin and 16 M Ca²⁺ were added (30 min). The perfusion medium was gassed continuously with 95 % O₂-5 % CO₂ and the temperature was maintained at 37 °C. Following the collagenase treatment, the tissue was minced (tissue mincer, Bachofen, Germany) and incubated with the collagenase-containing perfusion buffer for an additional 15 min in a shaker (37 °C). The following steps were carried out at room temperature. After filtering through nylon gauze, the filtrate containing the isolated cells was washed twice with increasing [Ca²⁺] to reach, stepwise, a concentration of 0.5 mM. In a further step, the suspension was layered on 4 % bovine serum albumin (ICN Flow, Meckenheim, Germany) and 1 mM Ca²⁺-containing buffer and centrifuged for 2 min at 10 g. The cardiomyocyte pellet was washed three times in PBS and boiled immediately in 1 ml of SDS-PAGE sample buffer. Western blot analysis was performed as previously described (Gödecke et al. 1998). Quantification of eNOS bands was performed by scanning using a Densitometer (Molecular Dynamics).

Total RNA was isolated from whole hearts or cardiac myocytes by the LiCl-urea method (Auffray & Rougeon, 1980). Reverse transcription of 5 g total RNA was carried out in a total of 50 l using MMLV reverse transcriptase (Life Technologies, Eggenstein, Germany). RT products were serially diluted 1:3.1 and 2 l each of the RT reaction and the dilution steps were used for PCR using either eNOS- or angiotensin-converting enzyme (ACE)-specific primers (1 min at 94 °C, 2 min at 55 °C, 2 min at 72 °C, 40 cycles). Primer sequences were eNOS: 5'-CTG GAC ATC ACT TCC CCG-3' and 5'-GAG CTG GCT CAT CCA CGT-3'; ACE: 5'-CAA TTA CCT GCT AAA GAT GGC C-3' and 5'-TAC TCC AGC AGT GCC TTG G-3'.

Measurement of cAMP/cGMP

Langendorff perfused hearts from WT or eNOS-/- mice were allowed to equilibrate for 30 min under constant flow. For the determination of basal levels of cyclic nucleotides, hearts were freeze clamped after this period. To determine the cAMP/cGMP levels under -adrenergic stimulation, maximal dobutamine concentrations were applied (300 nM) until a steady state in LVP was reached (4-6 min). Then hearts were snap frozen. Tissues were stored at -80 °C. For the isolation of cAMP/cGMP, frozen tissues were weighed, and homogenized in a 10-fold volume of ice-cold 6 % trichloroacetic acid. Precipitated proteins were removed by centrifugation (10 min, 7500 r.p.m., 4 °C in an Eppendorf microfuge). Supernatants were extracted four times with a 5-fold volume of water-saturated diethylether. The aqueous phase was dried down under vacuum. The determination of cAMP/cGMP was performed by ELISA (Amersham, Braunschweig, Germany) according to the manufacturer's instructions.

Isolated mouse hearts

Mice were injected I.P. with 250 U heparin and anaesthetized with urethane (1.5 g kg^-1 I.P.). The hearts were rapidly excised and transferred for preparation of the aortic trunk to warm, oxygenated Krebs-Henseleit buffer. The aorta was cannulated and heart was perfused in a non-recirculating Langendorff mode at constant flow (7 kPa, i.e. 70 cmH2O) with a modified Krebs-Henseleit buffer containing (mm): NaCl 116, KCl 4.6, MgSO4 1.1, NaHCO3 24.9, CaCl2 2.5, KH2PO4 1.2, glucose 10 and EDTA 0.5, equilibrated with 95 % O2 and 5 % CO2 (pH 7.4, 37 °C). Hearts were allowed to equilibrate for 30-40 min until a constant coronary flow was reached. Then perfusion was switched to constant volume using a roller pump. The pump rate was adjusted to result in a perfusion pressure of approximately 60 mmHg. For the analysis of -adrenergic stimulation flow was increased by a factor of two and after equilibration dobutamine was infused from a side arm.

Coronary flow was measured with a transit-time ultrasound flowmeter located above the aortic cannula (Transonics, Ithaca, NY, USA). Dobutamine and CaCl₂ were infused into the aortic cannula at infusion rates ranging from 0.2 to 1 % of coronary flow. LVP was recorded by insertion of a buffer-filled latex balloon connected to a Statham P23XL pressure transducer into the left ventricle. The volume of the balloon was adjusted to induce an end-diastolic pressure of 5 mmHg.

Determination of -adrenergic receptor density

The preparation of plasma membranes for the measurement of -adrenergic receptor density has been described (Ihl-Vahl et al. 1995). In brief, heart and lung tissues were homogenized in 35 ml buffer A containing (mM): Tris-HCl 50, EDTA 5 and EGTA 2 (pH 7.2, 4 °C), using a Polytron (Kinematika, Lucerne, Switzerland; 10 000 r.p.m., 310 s). The proteinase inhibitors benzamidine (1 mM) and PMSF (1 mM) were used in all buffers. After sedimentation (350 g, 10 min, 4 °C) the supernatant was filtered through two layers of cheese cloth. Plasma membranes were collected by centrifugation (48 000 g, 10 min, 4 °C) and washed twice in 35 ml buffer A by centrifugation (48 000 g, 10 min, 4 °C). The final membranes were resuspended in 50 mM Tris-HCl (pH 7.9), to give a final protein concentration of 2 mg ml^-1 for heart membranes.

-Adrenergic receptor density was determined by radioligand-binding techniques using the radiolabelled antagonist of -adrenergic receptors [¹²⁵I]iodocyanopindolol ([¹²⁵I]Cyp) as a specific ligand (Engel et al. 1981; Ihl-Vahl et al. 1995). The number of -adrenergic receptors was determined in saturation experiments using increasing concentrations of [¹²⁵I]Cyp (0.02-0.320 nM) in an assay volume of 250 l including 20-30 g membrane protein per tube for heart membranes. The incubation (1 h, 30 °C) was stopped by rapid filtration (Whatman GF-C) with ice-cold buffer (3 4 ml, 50 mM Tris-HCl, pH 7.4).

Non-specific binding was determined by measuring the residual binding in the presence of alprenolol (10^-6 M) and accounted for up to 20 % of the total binding in heart, which is in good agreement with previously published results (Ihl-Vahl et al. 1995). The data were analysed using curve-fitting techniques with least-squares curve fitting based on the mass law action (GraphPad). Protein determination was performed according to the method of Bradford using bovine serum albumin as a standard.

Electrophysiology

Cell isolation. Mice (5-6 months old) were injected with heparin (5000 U kg^-1 I.P.) and killed after 30 min by cervical dislocation followed by exsanguination. The chest was opened and the aorta was cleaned of connective tissue. After cannulation with a plastic cannula (0.8 mm internal tip diameter), hearts were mounted in a perfusion system (constant flow 2 ml min^-1, 37 °C); the first perfusion with Ca²⁺-free solution started less than 2 min after killing. After 3 min of equilibration, enzyme medium was perfused for approximately 15 min. Then enzymes were washed out with KB medium (5 min), and the heart was disconnected from the perfusion system. In KB medium, the ventricles were cut into small chunks and gently triturated. The resulting cell suspension was filtered and stored in KB solution (22 °C) until use (3-7 h).

Composition of solutions. Ca²⁺-free solution (mM): 118 NaCl, 4 KCl, 1 MgCl₂, 1.6 NaHPO₄, 24 NaHCO₃, 5 pyruvic acid, 20 taurine and 10 glucose, pH 7.4. All Ca²⁺-free solutions were gassed with 95 % O₂- 5 % CO₂. Enzyme medium: Ca²⁺-free solution supplemented with 20 M CaCl₂, 0.2 mg ml^-1 collagenase (Type II, Worthington, lot M8j2151, 225 units mg^-1), 1 mg ml^-1 bovine serum albumin (Sigma). KB medium (mM): 50 L-glutamic acid, 30 KCl, 3 MgSO₄, 0.1 EGTA, 30 KH₂PO₄, 10 Hepes, 20 taurine and 10 glucose, pH 7.3. Tyrode solution (synonymous with PSS): NaCl 150, KCl 5.4, CaCl₂ 1.8, MgCl₂ 1.2, glucose 10 and Hepes-NaOH 5, adjusted to pH 7.4. Internal pipette solution: 140 CsCl, 5 NaCl, 0.5 MgCl₂, 10 Hepes and 0.005 EGTA, pH 7.3.

Voltage clamp. Whole-cell membrane currents were recorded with patch pipettes of approximately 3 M resistance using an RK-300 patch-clamp amplifier (Biologic, Echirolles, France). Currents were filtered at 1 kHz and not corrected for leakage. On-line, they were recorded with a thermo-writer (Gould, TA 550, Cleveland, OH, USA). A microcomputer in combination with a CED-1401 interface (CED, Cambridge, UK) was used for generating the voltage-clamp commands and for storing the digitized data (digitizing frequency 2 kHz). To make sure that cell dialysis was not a severe problem, experiments were repeated with nystatin-perforated instead of ruptured patches; within the experimental time of 5-10 min, the comparison did not reveal peculiarities (4 cardiac myocytes from eNOS-/- mice).

Statistics

Data for repeated measures were analysed using two-way ANOVA followed by a post hoc Bonferroni test to analyse statistical significance of single data points using Prism 3.0 software (GraphPad).

	RESULTS

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eNOS expression in murine cardiac myocytes

To analyse whether eNOS is also expressed by murine cardiac myocytes, equal amounts of protein extracts from a WT heart and purified cardiac myocytes were analysed by Western blotting (Fig. 1A). As a negative control, extracts of eNOS-/- hearts were also loaded on the gel. eNOS-specific bands were detected in extracts from WT hearts and cardiac myocytes but not in extracts derived from eNOS-/- hearts. Densitometric analysis of three independent experiments revealed that approximately 20 % of the cardiac eNOS protein was detectable in the fraction of cardiac myocytes. nNOS- and iNOS-specific antibodies failed to detect any specific band.

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Figure 1. Expression analysis of eNOS in the murine heart A, Western blot of 150 g protein extracts from a WT and eNOS-/- heart and of isolated cardiac myocytes (CM) from a WT heart. The eNOS-specific band is indicated. B, RT-PCR analyses of eNOS expression in WT heart and isolated cardiac myocytes. Serial dilutions of cDNA were subjected to PCR reactions as described in Methods using primers specific for eNOS or ACE. Reactions were performed on cDNA derived from 200 ng (1) to 2 ng (0.01) of total RNA. The dilution factors are indicated below each lane.

These results were verified by estimation of the extent of endothelial cell contamination in the cardiac myocyte fraction as follows. RT-PCR analysis of eNOS and ACE expression in heart and cardiac myocytes was performed. As shown in Fig. 1B, amplification of eNOS and ACE transcripts in total RNA from the whole heart resulted in amplification products with an intensity ratio of approximately 1:1. In the myocyte fraction, however, the detection limit of eNOS-specific signals was three times lower compared with the ACE-specific bands. Since eNOS mRNA accumulates to higher levels than ACE mRNA in the myocardial fraction, these data strongly suggest that the eNOS expression found is cardiomyocyte specific.

eNOS attenuates the inotropic response to -adrenergic stimulation

The effects of eNOS disruption on the development of left ventricular force under -adrenergic stimulation were analysed in isolated perfused hearts. In order to exclude chronotropic effects of -adrenergic stimulation, hearts were paced at 600 beats min^-1, a frequency slightly above the maximal frequency that was obtained with dobutamine in unpaced hearts (data not shown). As shown in Fig. 2, intracoronary application of dobutamine (10-300 nM) dose-dependently increased LVP in WT hearts from 94 mmHg by 36 mmHg at 300 nM dobutamine. This was accompanied by an increase of dP/dt_max from basal (3700 mmHg s^-1) by 78 %. In eNOS-/- hearts the basal contractile parameters were slightly but not significantly higher (LVP: 111 ± 11 mmHg; dP/dt_max: 4493 ± 320 mmHg s^-1; n = 6), the dose-response curve was steeper and the maximal inotropic response was significantly higher (LVP: +50 mmHg; dP/dt_max: +82 %; n = 6). Coronary perfusion pressure decreased with higher -adrenergic stimulation. However, no differences between WT and eNOS-/- hearts were found.

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Figure 2. Inotropic response of WT and eNOS-/- hearts to -adrenergic stimulation The dose-response curves of LVP and dP/dtmax elicited by dobutamine (10-300 nM) for WT () and eNOS-/- () are shown. Symbols indicate means ± S.D. for n = 6 experiments. ** Significant difference between groups by two-way ANOVA followed by Bonferroni post hoc test (P < 0.01).

A possible mechanism by which NO could modulate -adrenergic stimulation might be a decrease in cAMP via activation of the cGMP-stimulated phosphodiesterase (PDEII). Therefore, the effect of the PDEII inhibitor MEP2 (NPT 15392, 9-hydroxynonyl-hypoxanthine; 20 M) (Coffey et al. 1984) on the contractile force of WT and eNOS-/- hearts was analysed (n = 4). Inhibition by PDEII affected neither basal LVP and dP/dt, nor the inotropic response to maximal -adrenergic stimulation (data not shown). In addition, cardiac cAMP and cGMP levels were determined under basal conditions and after maximal -adrenergic stimulation (300 nM dobutamine). In WT hearts cAMP levels increased from 403 ± 126 to 772 ± 97 fmol (mg protein)^-1 (P < 0.01; n = 6). cAMP levels in eNOS-/- hearts were not different from the values found in WT hearts (476 ± 193 fmol (mg protein)^-1 basal vs. 663 ± 101 fmol (mg protein)^-1 with 300 nM dobutamine; n = 6). No significant differences in cGMP levels between WT and eNOS-/- hearts were detectable under all conditions analysed (WT: 275 ± 57 fmol (mg protein)^-1 basal vs. 306 ± 111 fmol (mg protein)^-1 with 300 nM dobutamine; eNOS-/-: 256 ± 114 fmol (mg protein)^-1 basal vs. 244 ± 91 fmol (mg protein)^-1 with 300 nM dobutamine; n = 6 in each group).

To explore whether eNOS-/- disruption resulted in a change of Ca²⁺ dependency, hearts were perfused with medium containing increasing concentrations of Ca²⁺ (1.5-4.5 M). As shown in Fig. 3, elevation of extracellular [Ca²⁺] resulted in a significant increase of LVP and dP/dt_max in WT and eNOS-/- hearts. The observed changes in contractile parameters were not different between WT hearts and eNOS-/- hearts.

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Figure 3. Inotropic response of WT and eNOS-/- hearts to elevated [Ca2+] The curves show the inotropic response of WT () and eNOS-/- hearts () to increased extracellular [Ca2+] (1.5-4.5 mM). Symbols indicate means ± S.D. for n = 6 experiments. n.s., no significant difference between WT and eNOS-/-.

In a separate series of experiments we analysed whether changes at the level of -adrenergic receptors might be involved in the augmented inotropic response of eNOS-deficient hearts. For this purpose -adrenergic receptor densities and affinities were determined in cardiac membrane preparations from WT and eNOS-/- hearts using [¹²⁵I]Cyp as a specific ligand. As shown in Fig. 4, -adrenergic receptor density in eNOS-/- hearts increased by 50 % in comparison to that of WT hearts (80 fmol (mg protein)^-1). The affinity for the ligand expressed as K_d, however, was not affected by the eNOS deletion. Similar results were obtained in lung tissue: in WT lung -adrenergic receptor density amounted to 680 ± 461 fmol (mg protein)^-1 and in eNOS-/- lungs receptor density was significantly elevated to 1243 ± 923 fmol (mg protein)^-1 (n = 6, P < 0.05). Again, K_d values were not altered. (WT: 119 ± 60 pM; eNOS-/-: 129 ± 131 pM; n = 6, not significant).

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Figure 4. Densities and affinities of -adrenergic receptors in WT and eNOS-/- hearts The density and affinity of -adrenergic receptors were determined in cardiac membrane preparations as described in Methods using [125I]Cyp as ligand. Bars indicate means ± S.D. of n = 6 experiments. , WT; , eNOS-/-. ** P < 0.01 vs. WT (ANOVA).

eNOS is not involved in transduction of the anti-adrenergic effects of ACh and adenosine

We then investigated whether eNOS-derived NO contributed to the anti-adrenergic effects of ACh and adenosine. Hearts were stimulated with 0.1 M dobutamine, resulting in elevated LVP and dP/dt in WT and eNOS-/- hearts. Adenosine (10 M) and ACh (100 nM) potently antagonized the dobutamine-induced increase of contractile function. From the data summarized in Fig. 5 it is evident that the two agonists significantly attenuated the dobutamine effect on contractile force to the same level. There were no significant differences in contractility between WT and eNOS-/- hearts after inhibition of the adrenergic effect by adenosine or ACh.

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Figure 5. The anti-adrenergic effect of ACh and adenosine Quantitative data demonstrating the anti-adrenergic effect of adenosine and ACh in WT and eNOS-/- hearts. Bars represent means ± S.D. for n = 6 experiments in each group. , WT; , eNOS-/-. ** P < 0.01 vs. WT (two-way ANOVA/Bonferroni post hoc test). † P < 0.01 vs. basal conditions (Student's unpaired t test). ‡ P < 0.01 vs. 0.1 M dobutamine (Student's unpaired t test).

Role of eNOS in the modulation of L-type Ca²⁺ channel current

To analyse the role of eNOS in the modulation of the L-type Ca²⁺ channel current, I_Ca,L was measured in voltage-clamp experiments. The density of I_Ca,L was 13.0 ± 1.1 pA pF^-1 in myocytes from WT and 13.3 ± 1.0 pA pF^-1 in myocytes from eNOS-/- hearts, the small difference not being significant (n = 13). Isoproterenol dose-dependently stimulated I_Ca,L in WT and eNOS-/- cells to a similar extent (Fig. 6), reaching a plateau at 100 nM isoproterenol. At saturating concentrations, the stimulation of peak I_Ca,L by isoproterenol was 172 ± 27 % in WT and 179 ± 27 % in eNOS-/- myocytes (not significant).

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Figure 6. Effect of -adrenergic stimulation on peak ICa,L in isolated cardiac myocytes , WT; , eNOS-/- cardiac myocytes. Data are normalized to peak ICa,L in the absence of isoproterenol. Data points represent means ± S.D. The differences between WT and eNOS-/- cardiac myocytes are not significant. For original current traces see Fig. 7A (WT) and C (eNOS-/-).

WT cardiac myocytes responded to dibutyryl-cGMP (50 M) with a significant reduction of basal I_Ca,L to 72 ± 12 % (n = 5) while eNOS-/- I_Ca,L was insensitive to cGMP (108 ± 11 %, n = 5). In contrast, dibutyryl-cGMP attenuated the I_Ca,L in prestimulated (30 nM isoproterenol) WT cells to 65 ± 15 % (n = 4) and in eNOS-/- cells to 63 ± 13 %. These effects were not significantly different.

The anti-adrenergic effect of carbachol on I_Ca,L was studied in another series of experiments (Fig. 7). In cells from WT mice, 30 nM isoproterenol caused I_Ca,L to increase from -2.1 to -3.7 nA, i.e. to 176 % (Fig. 7A). Subsequent application of 5 M carbachol on top of 30 nM isoproterenol reduced peak I_Ca,L within 5 min to -2.7 nA, which is 130 % of the control in the absence of both drugs Fig. 7B). In cardiac myocytes from eNOS-/- mice, 30 nM isoproterenol increased I_Ca,L from -3.4 to -5.2 nA (to 153 %; Fig. 7C), and subsequent addition of 5 M carbachol reduced the isoproterenol-stimulated I_Ca,L to -3.5 nA (103 % of basal I_Ca,L; Fig. 7D). The anti-adrenergic effect of carbachol reduced I_Ca,L along a time course that was indistinguishable between cells from WT and eNOS-/- mice (Fig. 7E).

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Figure 7. Anti-adrenergic effect of carbachol in isolated cardiac myocytes Anti-adrenergic effect of carbachol (5 M; CCh) on the isoproterenol (30 nM; ISO)-stimulated ICa,L in WT (A and B) and eNOS-/- (C and D) cardiac myocytes. Top traces, 140 ms pulses from -45 to 0 mV. E, time course of modulation of peak ICa,L by Iso (30 nM) and Iso plus CCh (5 M) in WT (top) and eNOS-/- (bottom) ventricular myocytes. Exposure to and wash out of drugs are marked by horizontal bars. Note that reduction of Iso-stimulated ICa,L by CCh follows a time course that is very similar in WT and eNOS-/- (when fitted by exponential functions, the exponents were 1.16 min in WT and 1.25 min in eNOS-/-).

On average, cells from WT mice (n = 7) responded to 30 nM isoproterenol with an increase of I_Ca,L to 181 ± 12 % of basal and to the combination of 30 nM isoproterenol plus 5 M carbachol with an increase to 101 ± 23 % (Fig. 8). In cells from eNOS-/- mice (n = 6), these values were 180 ± 15 and 100 ± 20 %, respectively (Fig. 8). In conclusion, significant differences in the anti-adrenergic effects of carbachol could not be established between myocytes from WT and eNOS-/- mice.

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Figure 8. Quantitative data for normalized peak ICa,L for WT (n = 7) and eNOS-/- (n = 6) cardiac myocytes Bars represent means ± S.D. , WT; , eNOS-/-. ** P < 0.01 vs. Iso. There were no significant differences between the respective values in WT and eNOS-/-.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

eNOS-/- mice were chosen in this study to analyse the role of eNOS in the modulation of myocardial contractility. Expression analysis revealed that eNOS in addition to its localization in the coronary and endocardial endothelium was also expressed in cardiac myocytes of mice. This study is the first to provide a quantitative estimate of the cellular distribution of eNOS: by Western blot and RT-PCR analysis we found that approximately 20 % of cardiac eNOS was associated with cardiac myocytes. The presence of eNOS in cardiomyocytes was previously reported for rat and human (Balligand et al. 1995; Seki et al. 1996; Wei et al. 1996). Thus, eNOS appears to be quantitatively sufficient to modulate myocardial contractility in a paracrine as well as autocrine manner in mice.

Western blot analysis of total cardiac protein extracts or isolated cardiac myocytes failed to detect other NOS isoforms. The relative abundance of these enzymes was very low in the murine heart and no massive upregulation of the other NOS isoforms occurred in eNOS-/- hearts. Similar conclusions were drawn by Vandecasteele et al. (1999) using a semi-quantitative RT-PCR to analyse expression of iNOS and nNOS in eNOS-deficient mouse hearts. Thus, phenotypical alterations in eNOS-/- hearts can be ascribed solely to the deletion of eNOS.

Analysis of myocardial contractility revealed similar basal contractile parameters such as LVP and dP/dt_max in WT and eNOS-/- hearts. In contrast, the inotropic response to catecholamines was significantly enhanced. This finding is consistent with data presented by Gyurko et al. (2000) who demonstrated no differences in basal contractility but elevated values under maximal -adrenergic stimulation in eNOS-/- hearts. However, similar to data obtained by Han et al. (1998) and Vandecasteele et al. (1999), we found no differences in the response to -adrenergic agonists of I_Ca,L in isolated cardiac myocytes from eNOS-/- hearts. These discrepant findings may be explained by assuming that the intrinsic activity of eNOS in cardiac myocytes was too low to influence I_Ca,L, while endothelium-derived NO in the intact heart acted in a paracrine manner to attenuate the response to -adrenergic stimulation. Several lines of evidence already support this conclusion. Experiments in cardiac myocytes using dibutyryl-cGMP showed that the isoproterenol-induced increase in I_Ca,L in WT and eNOS-/- cells was attenuated, demonstrating that the signal transduction pathway of NO was operative. A predominating paracrine mode of action of NO may also explain the results of Vandecasteele et al. (1999), who found an unaltered inotropic response to -adrenergic stimulation of papillary muscles taken from eNOS-/- hearts. Since flow-mediated generation of shear stress is known to be a key determinant of endothelial NO formation, this stimulus is certainly absent in papillary muscle preparations, but is present in a perfused organ such as the Langendoff heart. In addition, it is possible that the papillary muscles used were to some extent functionally compromised, since they tolerated a maximal pacing frequency of only 240 beats min^-1, which is far below the resting heart rate of mice and the pacing rate used in our study (600 beats min^-1). Whatever the mechanism may be, our data now clearly demonstrate that eNOS is the critical NOS isoform that down-regulates inotropic responsiveness to catecholamines. Our findings obtained from eNOS-/- hearts support results of earlier experiments in rat cardiac myocytes (Balligand et al. 1993) and canine and human hearts in situ (Hare et al. 1995a; Keaney et al. 1996) using NO synthase inhibitors.

The mechanism by which NO attenuates the inotropic response to -adrenergic stimulation may involve modulation of several components of the signal transduction cascade from the -adrenergic receptor to L-type Ca²⁺ channels (for review see Kelly et al. 1996). Our data provide clear evidence that the density of -adrenergic receptors is significantly elevated in eNOS-/- hearts. This finding is in contrast to those of Gyurko et al. (2000) who could not detect alterations of -adrenergic receptor levels in eNOS-/- hearts. The reason for these discrepant results is presently unclear but, whatever the explanation, several findings argue for only a minor contribution of -adrenergic receptor density to the increased sensitivity to catecholamines. (1) No differences in I_Ca,L were found either in basal or in catecholamine-stimulated isolated cardiac myocytes. (2) Inotropic changes were also found in WT hearts under acute NOS inhibition with L-NMMA (authors' unpublished results), which should not be accompanied by a receptor up-regulation via enhanced expression. Thus, up-regulation of -adrenergic receptors in eNOS-/- hearts seems to involve the vascular rather than the myocyte compartment. The finding of similar changes in the lung supports the validity of our data and suggests that eNOS is important in the control of -adrenergic receptor levels in various vascular beds. Alternatively, hypertension in eNOS-/- mice may be causally involved in -adrenergic receptor up-regulation. Provided this is a more general phenomenon, systemic upregulation of the -adrenergic receptor also in resistance vessels in eNOS-/- mice could result in a decrease in total peripheral resistance. This mechanism may potentially compensate for the loss of vasodilatory NO and thereby may limit hypertension in eNOS-/- mice.

It was demonstrated previously, that NO may act on components downstream of the Ca²⁺ channel (e.g. phospholamban, troponin-I), thereby modulating the Ca²⁺ sensitivity of cardiac myocytes (Shah et al. 1994; Bartel et al. 1995). It is also conceivable that given the Ca²⁺ dependence of eNOS, elevation of mean [Ca²⁺]_i should enhance NO formation in WT hearts only. However, experiments using the receptor-independent increase in [Ca²⁺]_i in the isolated heart suggest that the Ca²⁺ handling is not altered. Thus, NO seems to specifically target the -adrenergic signal transduction cascade between receptors and Ca²⁺ channels. In line with this finding is the observation that the basal I_Ca,L was not affected by the eNOS mutation.

Previous work suggested that NO in a cGMP-dependent manner may affect cAMP levels and Ca²⁺ currents (I_Ca,L) via regulation of the activity of the cGMP-stimulated (PDEII) and -inhibited (PDEIII) phosphodiesterases (Mery et al. 1993; Kojda et al. 1996). Since eNOS-/- hearts responded more sensitively to -adrenergic stimulation, loss of the cGMP-stimulated PDEII activity in eNOS-deficient hearts could have mediated this effect. However, we could not detect changes in contractility in hearts treated with the PDEII inhibitor MEP2 (Coffey et al. 1984), which makes a role for PDEII in the attenuation of the -adrenergic response in mouse hearts rather unlikely.

Besides the demonstration of an operative cGMP pathway in isolated cardiac myocytes, treatment of cells with dibutyryl-cGMP attenuated basal I_Ca,L in WT cells. In contrast, basal Ca²⁺ current of eNOS-/- cells was not subject to modulation by cGMP. This result was surprising because the anti-adrenergic effect of dibutyryl-cGMP, which is most probably mediated by modulation of phosphodiesterase activity or direct phosphorylation of L-type Ca²⁺ channels by cGMP-dependent kinase, was identical in WT and eNOS-/- cells. Whether the lack of effect of cGMP on basal I_Ca,L in eNOS-/- cells reflects a desensitization of one or both of these mechanisms in the absence of NO formation or indicates a new function of NO in the modulation of I_Ca,L is presently unclear.

The role of NO as a mediator of the anti-adrenergic effect of ACh has been addressed previously in different experimental models, such as isolated cardiac myocytes, papillary muscle, and also the canine heart in situ. However, the results obtained either supported (Balligand et al. 1993; Hare et al. 1995b; Han et al. 1996, 1998) or refuted (MacDonell et al. 1995; Kilter et al. 1995; Vandecasteele et al. 1999) a role for NO as a mediator of the anti-adrenergic effect. Strong support for a role of eNOS was recently presented by Han et al. (1998) using isolated cardiac myocytes from eNOS-/- mouse hearts. In their experiments the isoproterenol-induced activation of I_Ca,L was resistant to carbachol. Confusingly, Vandecasteele et al. (1999) recently demonstrated a preserved parasympathetic modulation of I_Ca,L in eNOS-/- cardiomyocytes. In general, major phenotypical differences in the analysis of a specific gene function in transgenic animals may result from differences in the targeting strategy used to create a specific mutation or from differences in the genetic background of the animals used. Neither argument can explain the discrepancies between the results of Han et al. (1998) and Vandecasteele et al. (1999) since the same strain of mice was used (Huang et al. 1995). Our data generated in the isolated heart and in isolated cardiomyocytes were derived from an independently generated eNOS knockout mouse (Gödecke et al. 1998). Here we clearly and consistently demonstrate by two independent experimental models that in adult mouse hearts eNOS does not mediate the anti-adrenergic effect of ACh and adenosine. We found no differences between WT and eNOS-/- hearts when measuring both contractile responses and I_Ca,L.

One possibility to explain previous inconsistencies may be related to age-dependent alterations of the parasympathetic signal transduction cascade. Han et al. (1998) used 2- to 3-month-old mice while those in the study of Vandecasteele et al. (1999) and ours presented here were 3-6 months of age. Hare & Stamler (1999) speculated that progressive development of compensating mechanisms may be responsible for the preserved anti-adrenergic effect of ACh in the 3- to 6-month-old mice. Still another explanation was recently suggested by Ji et al. (1999) who found that muscarinic modulation of myocardial L-type Ca²⁺ channels switches during progressive differentiation of embryonic stem cell-derived cardiac myocytes: whereas during early developmental stages ACh attenuated -adrenergic effects predominantly via the NO-cGMP- phosphodiesterase pathway, muscarinic receptors blocked cAMP formation via G_i-proteins at the level of adenylate cyclase in later stages representing adult cardiomyocytes.

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Acknowledgements

This work was supported by the Biologisch-Medizinisches Forschungszentrum (BMFZ) of the Heinrich-Heine Universität, Düsseldorf. We wish to thank S. Küsters for excellent technical assistance.

Corresponding author

A. Gödecke: Institut für Herz- und Kreislaufphysiologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstraße 1, Postfach 10 10 07, 40001 Düsseldorf, Germany.

Email: axel.goedecke{at}uni-duesseldorf.de

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