© 1998 by European Society of Cardiology
Copyright © 1998, European Society of Cardiology
An inhibitor of nitric oxide synthase does not increase contraction or β-adrenoceptor sensitivity of ventricular myocytes from failing human heart
Imperial College School of Medicine at the National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, UK
* Corresponding author. Tel.: +44-171-352-8121 ext. 3311; fax: +44-171-823-3392; e-mail: sian.harding@ic.ac.uk
Received 24 October 1997; accepted 21 April 1998
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Abstract |
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 Top Abstract 1 Introduction2 Methods3 Results4 DiscussionReferences |
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Objective: Nitric oxide (NO) has been implicated in the depression of cardiac function in human heart failure. Some reports have identified iNOS (inducible nitric oxide synthase) within the myocyte component of the failing human heart, and NO is known to decrease the contraction amplitude of isolated ventricular myocytes. We have treated myocytes from failing human ventricle with a NOS inhibitor, NG-monomethyl-L-arginine (L-NMMA), in an attempt to restore contractile function. Methods and Results: Myocytes were isolated from failing and non-failing human ventricles and their contraction amplitude was measured during superfusion (32°C, 1–2 mmol/l Ca2+) and electrical stimulation (0.1–2 Hz). The contraction amplitude of myocytes from failing hearts was depressed in a frequency-dependent manner. At 1 Hz, the contraction amplitude of cells from non-failing heart was 4.70±0.53% cell shortening (mean±SEM, n=13 subjects), compared with 2.18±0.27% (P<0.01, 11 patients) from patients with ischaemic heart disease (IHD) or 2.56±0.74% (P<0.02, six patients) with dilated cardiomyopathy (DCM). Superfusion with 0.1 mmol/l L-NMMA did not increase contraction amplitude in myocytes from failing heart at either 0.2 Hz (n=11) or 1 Hz (n=7). Responses to β-adrenoceptor stimulation were reduced in myocytes from failing human heart, with contraction amplitude in maximum isoprenaline 0.47±0.11 of that in high Ca2+ in the same cell (n=6), compared to 0.99±0.07 in non-failing heart (n=14, P<0.01). The presence of 0.1 mmol/l L-NMMA did not increase the isoprenaline/Ca2+ ratio in myocytes from failing heart (0.40±0.09, P=NS). Conclusion: These results do not suggest a functional role for tonic NO production in the frequency-dependent depression of contraction or β-adrenoceptor desensitisation in myocytes from failing human ventricle.
KEYWORDS Nitric oxide; Heart failure; Frequency; Contractility; Human
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Both muscle strips and single isolated myocytes from the left ventricle of failing human hearts exhibit reduced contraction in vitro, and do not display the ‘positive staircase’ (increase in contraction amplitude or force with increasing stimulation frequency) that is seen in non-failing myocardium [1–3]. Recently, the possibility has been raised that these observations may be explained by a negative inotropic effect of nitric oxide (NO), either produced within the myocyte or on the adjacent microvascular endothelium. Both of these cell types express eNOS (NOS3), the constitutive, Ca2+-activated nitric oxide synthase isoform that was first identified in endothelial cells [4]. In animal experiments, NO is capable of directly reducing contraction of cardiac myocytes [5], and can convert a positive to a negative staircase [6, 7]. The contraction of human myocytes or papillary muscle can also be reduced in the presence of a NO donor [8, 9]. Responses to β-adrenoceptor stimulation are particularly sensitive to the depressant effects of NO [9, 10].
Cardiac myocytes can also express iNOS (NOS2), the inducible, Ca2+-independent isoform, following exposure to cytokines [11]. Guinea-pigs in septic shock, which is accompanied by a marked induction of iNOS, yielded isolated myocytes with impaired contractile function. Contraction was significantly enhanced by NOS inhibitors, suggesting a role for tonic endogenous NO production by cardiac myocytes in the observed alterations [12]. The use of a washed, continuously superfused layer of freshly isolated myocytes precluded the involvement of other cell types in the generation of NO.
Inducible NO synthase activity, measured as the Ca2+-independent production of citrulline, has been detected within the myocardium of patients with idiopathic dilated cardiomyopathy (DCM) [13, 14], and iNOS protein was later shown to be localised to the myocyte cells [15]. This has led to the suggestion that, as with septic shock, production of NO within the cardiac myocyte itself results in the tonic depression of contraction observed in these patients. Inhibition of NOS with NG-monomethyl-L-arginine (L-NMMA) in vivo potentiated the contractile response to dobutamine in subjects with left ventricular dysfunction [16], implying that NO production could also contribute to the β-adrenoceptor desensitisation of heart failure. Myocytes isolated from dogs with pacing-induced heart failure had a depressed response to β-adrenoceptor stimulation, and this was reversed by application of the NOS inhibitor L-NAME [17]. However, not all studies show iNOS in failing human myocardium: Thoenes et al. [18]could detect iNOS protein or cGMP production in ventricular myocardium from patients with septic shock, but not ischaemic heart disease (IHD) or DCM. iNOS mRNA was seen in a proportion of patients in another study, but the incidence did not differ between hearts from donors and those from patients with IHD or DCM [19].
Studies on whole myocardium are complicated by the presence of non-myocyte cells within the tissue. To test the hypothesis that endogenous NO production within the myocyte itself is responsible for the depression of contraction, we have attempted to reverse the depression of contraction of single ventricular myocytes isolated from failing human heart by incubation with the NO synthase inhibitor L-NMMA. This compound inhibits both iNOS and eNOS and was effective in reversing the depressed contraction in myocytes from septic animals [12]. The evidence presented here does not suggest that there is any tonic effect of endogenously generated NO on myocyte contraction at either low or high frequencies of stimulation, or in the presence of β-adrenoceptor agonists.
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2 Methods |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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2.1 Isolation of cardiac myocytes
Failing ventricular myocardium was obtained from explanted hearts at the time of transplant. Patient details and primary etiology are shown in Table 1. Non-failing left ventricle was obtained from donor hearts that were not used for technical reasons, or from biopsies (50–150 mg) taken from patients at the time of coronary artery surgery, as before [3]. The investigation conforms with the principles outlined in the Declaration of Helsinki. Myocytes were isolated as previously described [3, 20].
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For explants or large samples of non-failing heart from donors, a sample of the ventricle (average weight, 1 g) was cut into chunks of approximately 1 mm3, which were incubated at 35°C in 25 ml of a low calcium (LC) medium containing nitrilotriacetic acid (NTA) as a calcium buffer. The composition of the LC medium was (mmol/l): NaCl 120, KCl 5.4, MgSO4 5, pyruvate 5, glucose 20, taurine 20, HEPES 10 and NTA 5, bubbled with 100% O2. The pH was adjusted to 6.95 and the measured free [Ca2+] was 1–3 µmol/l. The medium was changed three times at three-minute intervals (12 min total). The chunks were then drained and transferred to LC without NTA and with 50 µmol/l calcium added with 4 U/ml Sigma type XXIV protease (pronase) at the same temperature for 45 min. The solution was shaken under an atmosphere of 100% oxygen throughout. Two further digests were undertaken using Sigma type V collagenase (1 mg/ml) with or without Sigma hyaluronidase (0.5 mg/ml). The cell suspension was then filtered through 300 µm gauze, to remove undigested tissue, and the myocytes were pelleted by gentle centrifugation.
For biopsies of non-failing heart, patients were selected with stable angina undergoing coronary artery surgery with normal systolic ventricular function (ejection fraction >60%), as defined by left ventricular angiography. Patients were excluded if they had a history of myocardial infarction. All patients gave informed consent and the procedure was approved by the ethical committee of the Royal Brompton National Heart and Lung Hospital. Following the institution of cardiopulmonary bypass, the heart was electrically fibrillated and, prior to the initiation of cardioplegia, a small biopsy of the left ventricular free wall was made with a number 11 blade. The average biopsy weight was 125 mg. There were no adverse effects from this procedure. The sample was incubated in ice-cold LC medium for 30 min, then cut into 400 µm slices using a vibratome (Micro Cut 1200, Energy Beam Sciences). The slices were placed in an ice-cold solution of protease (0.5 mg/ml) and collagenase (1.5 mg/ml) in LC solution, but with the addition of 50 µmol/l calcium and without NTA. This was then warmed to 35°C for 30 min whilst being gently shaken in an atmosphere of 100% oxygen. This solution was then exchanged for one containing collagenase (1.0 mg/ml) and hyaluronidase (0.5 mg/ml) and incubated for a further 90 min. The supernatant was removed and the myocytes were pelleted by gentle centrifugation before resuspension in LC without NTA.
2.2 Myocyte contraction studies
Myocytes were superfused at 32°C in Krebs-Henseleit solution of the following composition (in mmol/l): NaCl 119, KCl 4.2, MgSO4 0.94, KH2PO4 1.2, NaHCO3 25, glucose 11.5, bubbled with 95% O2 5% CO2, pH 7.4, and containing 2 mmol/l Ca2+. Cells were chosen for study on the basis of a number of criteria: (1) morphological appearance (rod-shaped, no large blebs or areas of hypercontracture), (2) sarcomere length >1.70 µm, (3) no spontaneous contractions when unstimulated at 2 mmol/l Ca2+ and (4) steady contraction amplitude and diastolic length at a stimulation rate of 0.2 Hz. Contraction amplitude was monitored with a video-based system running at either 50 Hz with a spatial resolution of 1 in 256, or at 100 Hz and 1 in 512. Following a stabilisation period of at least 10 min at 0.2 Hz, the stimulation frequency was increased to 1 Hz for 1–2 min, then returned to 0.2 Hz. L-NMMA, 0.1 mmol/l, was superfused onto the myocytes for 10 min, and the increase in frequency was repeated. Contraction was measured again after washout of L-NMMA. Myocytes were also superfused for 2–3 min with 8 mmol/l Ca2+, a concentration which will produce a contraction amplitude close to maximum for most cells [21]. Cumulative concentration–response curves to isoprenaline were constructed in 1 mmol/l Ca2+ (0.2 Hz) in the presence and absence of 0.1 mmol/l L-NMMA. The maximum was defined either as the point at which increasing concentrations of isoprenaline had no further effect on contraction amplitude, or when signs of toxicity were noted (arrhythmias, contracture). Experiments were used only if the inotropic or toxic effects of isoprenaline or Ca2+ were reversible on washout.
2.3 Statistical analysis
Results from up to three myocytes were pooled for each patient, so that n refers to patients. Comparisons were done either by paired t-test, or by one-way ANOVA followed by pairwise comparison of means using the Fisher test.
2.4 Materials
Salts were obtained from BDH/Merck (Poole, UK) and were of AnalaR grade, apart from glucose and KCl, which were used at AristaR grade for the low calcium solution. Isoprenaline HCl was obtained from Sigma (Poole, UK) and L-NMMA was from Calbiochem (Beaston, UK).
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3 Results |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Contraction amplitudes at 0.2 and 1 Hz stimulation rates are compared in Fig. 1 for myocytes from the left ventricle of non-failing, IHD or DCM hearts. As we have previously shown [3], there was a positive staircase in myocytes from non-failing hearts, with the amplitude being significantly greater at 1 than at 0.2 Hz (P<0.001), however, there was little change with frequency in cells from failing hearts. There was no difference between myocytes from donor hearts and biopsies with respect to their frequency dependence of contraction. Contraction amplitude at 1 Hz was significantly larger in myocytes from non-failing heart than in those from either IHD or DCM patients (ANOVA, P<0.001). Increasing the stimulation frequency above 1 Hz did not increase the amplitude of myocytes from either failing or non-failing hearts [3]. The differences in amplitude between myocytes from IHD and DCM patients were not statistically significant.
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There was little evidence of diastolic contracture in any group at either low or higher stimulation rates. Diastolic sarcomere lengths at 0.2 and 1 Hz were 1.84±0.02 and 1.81±0.03 µm, respectively, for non-failing (n=13, three donors and ten biopsies), 1.90±0.03 and 1.88±0.04 µm for IHD (n=11), and 1.86±0.08 and 1.84±0.08 µm for DCM (n=6).
In a subgroup of cells from failing hearts, amplitudes were compared at 0.2 and/or 1 Hz before and after superfusion with 0.1 mmol/l L-NMMA (Fig. 2). There was no increase in amplitude in the presence of L-NMMA at either frequency, and this was true for myocytes from patients with IHD (n=7), DCM (n=3) and aortic valve disease (n=1). One of the IHD samples was from right ventricle, the remainder were from left ventricle. For comparison, the responses of nine of the same myocytes to 8 mmol/l Ca2+ are shown. There was a pronounced increase in contraction amplitude with high Ca2+, indicating that the cells had not been contracting at their maximum capacity at the basal Ca2+ concentration.
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Nor did concurrent infusion of L-NMMA increase the responsiveness of the myocyte to β-adrenoceptor stimulation. Fig. 3a shows a myocyte from a DCM heart where consecutive concentration–response curves to isoprenaline were performed in the absence and presence of 0.1 mmol/l L-NMMA: clearly, the NOS inhibitor did not potentiate the β-adrenoceptor effect. A subsequent concentration–response curve for Ca2+ on the same cell is shown, and the maximum response to Ca2+ is much larger than that to isoprenaline. This isoprenaline–Ca2+ ratio of 0.38, which, together with the EC50 value of 52 nmol/l, shows that there was a severe degree of β-adrenoceptor desensitisation in this myocyte. In experiments on myocytes from seven failing hearts (three IHD and four DCM), 30 nmol/l isoprenaline increased the contraction amplitude from 1.08±0.45 to 4.06±1.49%. In the presence of 0.1 mmol/l L-NMMA, the same concentration of isoprenaline increased the amplitude from 1.03±0.46 to 3.40±1.19%. Neither basal nor isoprenaline-stimulated amplitude was significantly different from that in the absence of L-NMMA (paired t-test). Maximum isoprenaline–Ca2+ ratios were 0.47±0.11 in the absence and 0.40±0.09 in the presence of 0.1 mmol/l L-NMMA (n=6, P=NS, paired t-test). For comparison, Fig. 3b shows concentration–response curves to isoprenaline for myocytes from 14 non-failing left ventricles (seven donors and seven biopsies). The EC50 value for isoprenaline was 2.5±1.2 nmol/l and the maximum isoprenaline–Ca2+ ratio was 0.99±0.07 (P<0.01 compared to failing).
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The activity of the batch of L-NMMA was confirmed by an assay with lung tissue from LPS-treated rats, where Ca2+-insensitive NOS was reduced from 58 pmol citrulline/30 min to undetectable levels by incubation with 0.1 mmol/l L-NMMA.
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4 Discussion |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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The results from this study do not support the hypothesis that tonic production of endogenous NO within the myocytes of the failing heart contributes to the poor contraction observed in these cells. There was a clear contraction deficit in the myocytes from failing hearts used in the present study, since they showed no significant change in contraction amplitude between 0.2 and 1 Hz, compared with a marked increase in amplitude in cells from non-failing hearts. β-Adrenoceptor desensitisation was also evident, with the maximum contraction amplitude for isoprenaline being half that of high Ca2+ in myocytes from failing heart, while responses to isoprenaline and Ca2+ were equal in cells from non-failing ventricle. However, incubation of myocytes with the NO synthase inhibitor L-NMMA (0.1 mmol/l) before and during frequency increases or the addition of isoprenaline did not improve responses in the myocytes from failing heart.
This contrasts with our previous results in guinea-pigs with septic shock, where the same concentration of L-NMMA and duration of exposure was able to increase myocyte contraction amplitude by 47%, and so significantly reverse the depression of contraction [12]. L-NMMA at concentrations that were ten-fold lower than those used in the present study significantly alter the force–frequency relation of hamster myocardial strips [6]. We also confirmed the activity of the batch of L-NMMA used in the present experiments using lung tissue from LPS-treated rats. The human myocytes had not been working close to their limit, since high Ca2+ more than doubled the contraction amplitude; this means that the lack of effect of L-NMMA was not a consequence of the inability of the cells to contract further. Taking these controls into account, it is unlikely that the differences between myocytes from failing and non-failing hearts were caused by tonic NO production within the cells.
We could detect no significant difference between myocytes from IHD and DCM in the degree of contractile impairment at 1 Hz (this study), the extent of β-adrenoceptor desensitisation [20]or the slowing of relaxation [21]. In contrast, the induction of iNOS activity was reported to be associated primarily with DCM or inflammatory cardiomyopathies, but not with IHD or valve disease [14]. Similarly, the increase in protein levels of iNOS was found to be more pronounced in the myocytes of patients with DCM compared to IHD, although both were raised compared to non-failing myocardium [15]. However, other authors have reported patients with and without iNOS mRNA in both IHD and DCM [17, 19]and a similar pattern has been demonstrated for nitrate production [22]. Yet another group could not find any iNOS protein, or an increase in cGMP (a second messenger mediating the effect of NO), in patients with either DCM or IHD, although they found both iNOS protein and increased cGMP in samples from patients with septic shock [18]. Importantly, it has recently been shown that iNOS mRNA can be induced in human myocytes by incubation with cytokines but that iNOS protein levels are not increased [23]. This dissociation between mRNA and protein expression, which is found in many circumstances, may go some way towards explaining the discrepancies in the literature.
It may be suggested that, under other conditions, such as higher Ca2+, or at 37°C rather than 32°C, the effect of NO production within the myocyte does become important. However, we have shown that the frequency-dependent depression of contraction is quantitatively similar at 32 or 37°C, and at 2 mmol/l external Ca2+ or maximally activating external Ca2+ [3]. Furthermore, the degree of depression in myocytes is also quantitatively close to that seen in muscle strips from failing hearts, with contraction at 1–1.5 Hz being approximately halved compared to that in non-failing hearts [2]. This argues that there is little extra contribution of non-myocyte cells, such as endothelium (which could also produce NO), to the decrease in contraction in intact failing human myocardium.
A possibility that cannot be excluded is that the production of NO in the myocytes or endothelium in vivo has produced an irreversible change in the cells, which accounts for some or all of the contraction deficit seen. The mechanisms of β-adrenoceptor desensitisation have been relatively well defined, with decreased synthesis of β1-adrenoceptors, uncoupling of β2-adrenoceptors and increases in the inhibitory guanine nucleotide binding proteins [24]. Depression of the frequency response appears to be related to a decrease in activity of the sarcoplasmic reticulum Ca2+-ATPase [25, 26]. It will be interesting to see whether prolonged exposure to NO could produce changes in these proteins in normal hearts.
To summarise, our experiments argue against the current hypothesis that the frequency-dependent depression of contraction and β-adrenoceptor desensitisation in myocytes from failing human ventricle are directly related to tonic endogenous production of NO within the myocyte itself.
Time for primary review 40 days.
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Acknowledgements |
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This work was funded by the British Heart Foundation (PG/94116) and the Wellcome Trust. We are grateful to Professor Sir Magdi Yacoub and immunologists of Harefield Hospital, including Sean Allen, Rhoda McDouall, Chris Page and Chris Bravery for their generous assistance with the supply of human myocardium. We would like to thank Jane Mitchell, Thoracic Medicine, NHLI, for measuring iNOS activity in rat lung.
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Notes |
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1 Present address: Department of Cardiovascular Medicine, John Radcliffe Hospital, Oxford OX3 9DU, UK.
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References |
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