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Cardiovascular Research 2006 70(1):97-106; doi:10.1016/j.cardiores.2006.02.002
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Copyright © 2006, European Society of Cardiology

Are myocardial eNOS and nNOS involved in the β-adrenergic and muscarinic regulation of inotropy? A systematic investigation

Sarah R. Martin1, Krzysztof Emanuel1, Claire E. Sears, Yin-Hua Zhang and Barbara Casadei*

Department of Cardiovascular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, UK

* Corresponding author. Tel.: +44 1865 220132; fax: +44 1865 768844. Email address: barbara.casadei{at}cardiov.ox.ac.uk

Received 23 July 2005; revised 18 January 2006; accepted 1 February 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective The role of constitutive nitric oxide (NO) production in the regulation of β-adrenergic and muscarinic responses remains controversial. Conflicting data in left ventricular (LV) myocytes from eNOS knockout mice (eNOS– / –) have been ascribed to inconsistent experimental conditions (i.e., differences in the choice of controls, age of the mice, myocytes' stimulation frequency, and in the level of β-adrenergic stimulation); however, the recent identification of a neuronal-like NO synthase (nNOS) in the LV myocardium has raised the possibility that this isoform may be involved in the modulation of β-adrenergic and muscarinic responses.

Methods To address these issues we recorded sarcomere shortening at 35 °C under basal conditions, in the presence of isoproterenol (ISO, 10–100 nmol/L) and of ISO plus carbamylcholine (CCh, 1 µmol/L) in LV myocytes isolated from eNOS– / – and nNOS– / – mice, their wild type littermates (eNOS+/+ and nNOS+/+) or C57BL/6J mice. eNOS– / – and control myocytes were studied at 1 and 3 Hz, in the presence of 10 and 100 nmol/L ISO, and responses were compared between young (3 months) and old (leg12 months) mice.

Results Contraction did not differ between young eNOS– / – and eNOS+/+ mice at all stages of the experimental protocol, either at 1 or 3 Hz or in response to 10 or 100 nmol/L ISO. However, myocytes from old eNOS– / – mice showed a reduced inotropic response to ISO compared with age-matched eNOS+/+ mice (P=0.02). Similarly, there was a significant difference in the ISO response between eNOS+/+ and C57BL/6J myocytes (P<0.01), suggesting that experimental variables such as age and the choice of control animals may have contributed to the inconsistency in the results reported in the literature. In contrast, nNOS– / – myocytes showed greater contraction and slower relaxation at all stages of the experimental protocol (P=0.0003 and P=0.01 vs. nNOS+/+ myocytes).

Conclusions Constitutive eNOS expression in murine LV myocytes is not essential for the muscarinic-mediated inhibition of β-adrenergic signalling and does not appear to play a significant role in the regulation of basal and β-adrenergic myocardial contraction. Our data suggest that nNOS is the myocardial constitutive isoform responsible for the NO-mediated autocrine regulation of myocardial inotropy and relaxation.

KEYWORDS Nitric oxide; β-Adrenergic; Cholinergic; Inotropy; Autocrine; nNOS; eNOS; Age; Wild type controls


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
It is now well established that both endothelial (eNOS) and neuronal (nNOS) nitric oxide synthases are present in LV myocytes [1–3] but the relative contribution of these enzymes in the autocrine regulation of β-adrenergic inotropy remains a matter of debate. For instance, selective eNOS gene deletion (eNOS– / –) enhances the inotropic response to β-adrenergic stimulation in vivo and in isolated hearts [4–6] (Table 1), however all studies [5,9,10] except one [6] reported no difference between eNOS– / – and controls when the β-adrenergic inotropic response was evaluated in LV myocytes. Similarly, the basal and ISO-stimulated ICa in LV myocytes from eNOS– / – mice has not been found to differ from control mice [9–11]. In contrast, we have shown that both contraction and ICa are greater in LV myocytes from nNOS– / – mice both under basal conditions and in response to 2 nM ISO [12,13], suggesting that myocardial nNOS may play a previously unrecognised role in the regulation of myocardial function and Ca2+ handling [14].


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  		Table 1 Experimental conditions and choice of controls in previous studies in eNOS– / – mice

 
Similar discrepancies surround the role of myocardial constitutive NO production in the inotropic response to muscarinic receptor stimulation. As nNOS was first localised to the sarcoplasmic reticulum (SR)[2], it seemed unlikely that this isoform would be coupled to muscarinic cholinergic receptors in caveolae [15,16]. However, more recent data have indicated that nNOS may also be targeted to the sarcolemma [3] where it might conceivably be activated upon stimulation of β-adrenergic or muscarinic receptors. This hypothesis, however, has yet to be tested. Conversely, muscarinic signalling in LV myocytes of eNOS– / – mice has been studied extensively. In 1998, Han et al. [9] showed that muscarinic cholinergic inhibition of β-adrenergic contraction and ICa was significantly attenuated in LV myocytes from eNOS– / – mice. However, more recent work has indicated that the CCh-mediated inhibition of β-adrenergic signalling in the heart is unaltered in the presence of a targeted deletion of the eNOS gene [5,10,11].

How can this controversy in the literature be explained? It has been pointed out that conflicting data in eNOS– / – mice may be ascribed, at least in part, to inconsistent experimental conditions [14,17,18]. As illustrated in Table 1, investigators have employed different (i) control mice; (ii) frequency of stimulation of LV myocytes; (iii) levels of β-adrenergic stimulation; (iv) temperatures at which experiments were conducted and (v) have studied mice of different ages, raising the possibility that adaptive processes or the development of LV hypertrophy may have confounded the eNOS– / – myocardial phenotype (reviewed in [18]).

In view of these uncertainties, it remains unclear whether myocardial constitutive NO production plays a significant role in the autocrine regulation of β-adrenergic inotropy and muscarinic signalling in murine LV myocytes. We have addressed this issue by taking a two-sided approach: (i) we have systematically evaluated whether inconsistent experimental conditions (Table 2) may account for some of the controversial findings in eNOS– / – myocytes and (ii) we have investigated whether myocardial nNOS-derived NO modulates the inotropic and lusitropic response to β-adrenergic and cholinergic receptor stimulation in murine LV myocytes.


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  		Table 2 Experimental conditions used to elucidate the role of nNOS-derived and eNOS-derived NO in β-adrenergic and cholinergic myocardial contraction and to systematically investigate the role of potential confounders of previous published work

 

   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Animals
Mice homozygous for targeted disruption of the eNOS gene (B6.129P2-Nos3tm1Unc, eNOS– / – [19]) were purchased from Jackson Laboratories, crossed with C57BL/6J mice (Harlan, UK) and the eNOS– / – offspring of heterozygous mice was compared with their age-matched (2–4 months old or leg12 months old) wild type siblings (eNOS+/+) or with C57BL/6J mice (Harlan, UK). Young (2–4 months old) nNOS– / – mice [20] and their wild type siblings (nNOS+/+) were obtained from our established colony.

All protocols were in accordance with the Home Office Guidance on the Operation of Animals (Scientific Procedures) Act, 1986 (H.M.S.O.) and National Institute of Health (Guide for the Care and Use of Laboratory Animals).

2.2 Measurements of sarcomere shortening
LV myocytes were isolated using a standard enzymatic dispersion technique [12] and used within 8 h of isolation. Myocytes were perfused with a modified Tyrode's solution (in mM: 130 NaCl, 5.4 KCl, 1.2 MgCl2, 1.4 CaCl2, 5 HEPES, 10 glucose) and stimulated to contract at 1 Hz or 3 Hz with a 5ms square pulse using platinum electrodes inserted into the perfusion chamber. Sarcomere length was measured using the IonOptix SarcLenTM acquisition system. Only cells with well-defined membranes and striations and with stable and symmetrical contraction were included.

Sarcomere shortening was expressed as a percentage of basal sarcomere length (average length in µm: 1.79±0.006 in nNOS+/+, 1.77±0.009 in nNOS– / –, 1.79±0.007 in eNOS– / – and 1.78±0.008 in eNOS+/+ mice). The rate of sarcomere re-lengthening was evaluated by the time to 50% relaxation (TR50). All experiments were performed at 35±1 °C.

2.3 Experimental protocol
Following stabilization of contractile parameters, the perfusion solution was changed to one containing ISO (10–100 nmol/L, Sigma). After stabilization of the response to ISO, the solution was rapidly switched to one containing CCh (1 µmol/L, Sigma) and ISO. The bath was then washed out with Tyrode until the contraction returned to the initial basal values. Measurements from at least five steady-state contractions were averaged for each intervention. To elucidate the role of eNOS- and nNOS-derived NO in β-adrenergic inotropy and its CCh-mediated inhibition and to assess the impact of potential experimental confounders the protocol described above was carried out under the conditions listed in Table 2.

2.4 Statistics
Data are expressed as mean±S.E. Two-way repeated measures ANOVA was used to test between-groups differences in cell shortening and time to 50% relaxation (TR50) at each stage of the experimental protocol. Post-hoc analysis was carried out by using the Fishers' PLSD test. The response to ISO or CCh was evaluated by comparing absolute differences in percent sarcomere shortening or TR50 from baseline and ISO-stimulated contraction, respectively. The null hypothesis was rejected at P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Does myocardial constitutive NO production modulate β-adrenergic contraction and its inhibition by CCh?
To answer this question we compared the inotropic response to 100nmol/L ISO and 1 µmol/L CCh in the presence of ISO in LV myocytes from nNOS+/+ and nNOS– / – mice (n=37 and 38 cells, respectively) and eNOS+/+ and eNOS– / – mice (n=14 and 12 cells respectively).

LV myocytes from nNOS– / – mice showed a significantly greater shortening at each stage of the experimental protocol (Fig. 1, ANOVA P=0.0003), however, the increase in sarcomere shortening in response to ISO did not differ significantly between groups (7.97±0.44% in nNOS+/+ vs. 8.82±0.51% in nNOS– / –, P=0.21), indicating a parallel upward shift in contraction in nNOS– / – LV myocytes. The negative inotropic response to CCh was similar in the two groups (the difference from % sarcomere shortening in ISO was – 3.40±0.38 in nNOS+/+ vs. – 3.33±0.38 in nNOS– / –, P=0.89). Similarly, relaxation (TR50) was prolonged in nNOS– / – myocytes at all stages of the experimental protocol (ANOVA, P=0.01). TR50 decreased significantly in response to ISO in both groups (P<0.001); however, there was no difference in the TR50 response to ISO (P=0.19) or CCh (P=0.35) between nNOS– / – and nNOS+/+ myocytes (Table 3).


Figure 1
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  		Fig. 1 Role of nNOS on β-adrenergic and cholinergic inotropic responses in isolated LV myocytes. A: Examples of sarcomere shortening under basal conditions, in the presence of ISO (100 nmol/L), ISO+CCh (1 µmol/L) and after wash-out in LV myocytes from nNOS– / – mice and their wild type littermates (nNOS+/+) field-stimulated at 1 Hz. B: Average percent sarcomere shortening in LV myocytes from nNOS– / – and nNOS+/+. Note that shortening is significantly greater in nNOS– / – myocytes at all stages of the experimental protocol (*P<0.05 vs. nNOS+/+, n=37 cells per group).

 

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  		Table 3 Time to 50% relaxation (TR50, ms) in field-stimulated LV myocytes (1 Hz, unless indicated otherwise) under basal conditions, in the presence of isoproterenol (ISO, 100 nM, unless indicated otherwise) and ISO+carbamylcholine (CCh, 1 µM)

 
Basal sarcomere shortening and TR50 and their response to ISO and CCh did not differ between eNOS+/+ and eNOS– / – mice at any stage of the experimental protocol (ANOVA, P=0.90 and P=0.75, respectively; Fig. 2, Table 3).


Figure 2
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  		Fig. 2 The influence of mice genetic background on β-adrenergic and cholinergic inotropic response in LV myocytes isolated from eNOS– / – mice, their wild type littermates (eNOS+/+) and C57BL/6J mice. A: Examples of sarcomere shortening under basal conditions, in the presence of ISO (100 nmol/L), ISO+CCh (1 µmol/L) and after wash-out in LV myocytes (1 Hz) from eNOS– / – mice, their wild type littermates (eNOS+/+) and C57BL/6J mice. B: The line graph shows no differences in the average sarcomere shortening in LV myocytes from eNOS– / – (n=12) and eNOS+/+ (n=14) at each state of the experimental protocol. However, LV myocytes from C57BL/6 mice (n=20) showed a significant reduction in the inotropic response to ISO compared with eNOS+/+ myocytes (*P=0.007).

 
Taken together these findings indicate that nNOS-derived NO regulates basal myocardial contraction and relaxation but does not significantly influence the inotropic and lusitropic response to 100 nM ISO. Under the experimental conditions employed in this protocol (1 Hz stimulation frequency at 35 °C and using wild-type littermates as controls) we could not detect any effect of eNOS-derived NO on basal and β-adrenergic contraction and relaxation or on the CCh-mediated inhibition of β-adrenergic inotropy.

3.2 Could the choice of control animals affect these findings?
C57BL/6J mice are regarded as acceptable controls for the eNOS– / – mice, as the latter have been backcrossed onto the C57BL/6J background 8 times (see: http://jaxmice.jax.org/jaxmice-cgi/jaxmicedb.cgi?objtype=pricedetail and stock=002684). To test whether eNOS+/+ and C57BL/6J mice can be regarded as equivalent controls for the eNOS– / – mice, we compared basal contraction and the inotropic responses to ISO and CCh in LV myocytes from age-matched eNOS+/+ (n=14 cells) and C57BL/6J mice (n=20 cells).

Analysis of variance highlighted an overall difference in contraction between eNOS+/+ and C57BL/6J myocytes and a significant interaction between genotype and protocol (P<0.005). Basal shortening did not differ significantly between LV myocytes from eNOS+/+ and C57BL/6J mice (P=0.44, Fig. 2); however LV myocytes from eNOS+/+ had a significantly greater inotropic response to ISO compared to C57BL/6J myocytes (the difference from basal sarcomere shortening was 11.61±1.05% in eNOS+/+ vs. 8.30±0.41% in C57BL/6, P<0.005). Similar differences were observed between C57BL/6J and eNOS– / – myocytes. In contrast, CCh inhibition of ISO-stimulated inotropy did not differ between eNOS+/+ and C57BL/6J myocytes (– 6.03±0.92% in eNOS+/+ and – 5.30±0.76% in C57BL/6J, P=0.55). As shown in Table 3, TR50 was significantly prolonged in C57BL/6J myocytes compared with eNOS– / – and eNOS+/+ myocytes, both under basal conditions and in the presence of ISO or ISO+CCh (ANOVA, P<0.005); however, the lusitropic response to ISO did not differ between groups (P=0.37).

In summary, our findings indicate that relaxation and the inotropic response to ISO differ significantly between eNOS+/+ and C57BL/6J myocytes, suggesting that the latter may not be an appropriate control for eNOS– / – mice.

3.3 Does stimulation frequency affect the inotropic response to ISO and ISO+CCh?
To investigate whether the inotropic effects of myocardial NO production may become more apparent at higher stimulation frequencies, as suggested by Kaye et al. [21], we compared myocytes from eNOS– / – and eNOS+/+ mice stimulated at 1 Hz and 3 Hz.

Basal sarcomere shortening (Fig. 3) and TR50 (Table 3) did not differ significantly between cells stimulated at 1 Hz and at 3 Hz in either group (P=0.50 and 0.33 for eNOS+/+ cells and P=0.28 and 0.11 for eNOS– / – cells, respectively; n=12–14 cells in each group); however, the inotropic and lusitropic response to ISO was significantly larger at 1 Hz than at 3 Hz in eNOS+/+ myocytes (increase in % shortening: 11.61±1.01 at 1 Hz vs. 7.88±1.05 at 3 Hz, P<0.05; reduction in TR50 in ms: 8.06±1.15 at 1 Hz vs. 2.74±1.08 at 3 Hz, P<0.05). Similar findings were obtained in myocytes from eNOS– / – mice (15.26±0.85% at 1 Hz vs. 13.25±0.99% at 3 Hz, P=0.07 and 9.74±2.22 ms at 1 Hz vs. 4.72±1.60 ms at 3 Hz, P<0.05). There was no significant difference in the effect of CCh on ISO-stimulated contraction and relaxation between genotypes and stimulation frequencies.


Figure 3
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  		Fig. 3 Effect of stimulation frequency on β-adrenergic and cholinergic inotropic response in LV myocytes isolated from eNOS– / – mice and their wild type littermates (eNOS+/+). A: Examples of sarcomere shortening under basal conditions, in the presence of ISO (100 nmol/L), ISO+CCh (1 µmol/L) and after wash-out in LV myocytes from eNOS– / – mice and their wild type littermates (eNOS+/+) field-stimulated at 3 Hz. B: Average sarcomere shortening data in myocytes stimulated at 1 Hz (from Fig. 2) or 3 Hz show that the inotropic response to ISO was smaller at 3 Hz both in eNOS– / – (*P=0.07, n=12–13) and eNOS+/+ LV myocytes (*P=0.02, n=12–14).

 
3.4 Are differences in cell shortening between eNOS– / – and eNOS+/+ myocytes dependent on the level of β-adrenergic stimulation?
To evaluate whether the use of a near-maximal stimulating concentration of ISO (i.e., 100 nM) may have precluded further inotropic potentiation in eNOS– / – LV myocytes, we repeated our protocol using 10 nmol/L ISO in LV myocytes from eNOS+/+ and eNOS– / – mice field stimulated at 1 Hz (n=22 and 23 cells, respectively). As shown in Fig. 4 and Table 3, no significant differences were found between eNOS– / – and eNOS+/+ cell shortening and TR50 at all stages of the experimental protocol (ANOVA P=0.35 and P=0.68, respectively). The difference in sarcomere shortening in response to 10 nM ISO was 4.53±0.81% in eNOS+/+ myocytes and 4.09±0.74% in eNOS– / – (P=0.69). Similarly, the CCh-induced decrease in ISO-stimulated contraction was not different between groups.


Figure 4
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  		Fig. 4 Level of β-adrenergic stimulation and the inotropic responses of LV myocytes from eNOS+/+ and eNOS– / – mice. A: Examples of sarcomere shortening under basal conditions, in the presence of ISO (10 nmol/L), ISO+CCh (1 µmol/L) and after wash-out in LV myocytes from eNOS– / – mice and their wild type littermates (eNOS+/+) field-stimulated at 1 Hz. B: As shown with a higher ISO concentration (Fig. 2), there were no significant differences between eNOS– / – and eNOS+/+ sarcomere shortening in response to ISO (10 nmol/L) and ISO+CCh (n=9–10).

 
3.5 Does age affect the inotropic response to ISO and ISO+CCh in LV myocytes from eNOS– / – and eNOS+/+ mice?
To evaluate whether age differentially affects β-adrenergic and muscarinic responses in the presence or absence of eNOS, we compared LV myocytes isolated from young (2–4 months old) and old (leg12 months old) eNOS– / – and eNOS+/+ mice. Analysis of variance of cell shortening data highlighted a significant interaction between age and protocol (P<0.0005), which was driven by a significantly smaller β-adrenergic contraction in older mice (P=0.01 between myocytes from old and young eNOS+/+ mice and P=0.006 between myocytes from old and young eNOS– / – mice, Fig. 5). The negative inotropic effect of CCh was inhibited in older mice from both groups (P<0.05 between old eNOS– / – and eNOS+/+ myocytes and their respective younger controls).


Figure 5
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  		Fig. 5 Effect of age on β-adrenergic and cholinergic inotropic response in LV myocytes isolated from eNOS– / – mice and their wild type littermates (eNOS+/+). A: Examples of sarcomere shortening under basal conditions, in the presence of ISO (100 nmol/L), ISO+CCh (1 µmol/L) and after wash-out in LV myocytes from young (2–4 months old, as in Fig. 2) and old (leg12 months old) eNOS– / – and eNOS+/+ mice. B: Older age was associated with a suppressed inotropic response to ISO (*P=0.0004 between young and old eNOS– / – and P=0.006 between young and old eNOS+/+) and CCh (*P=0.06 between young and old eNOS– / – and P=0.008 between young and old eNOS+/+ mice). Furthermore, the increase in contraction in response to ISO was significantly more suppressed in old eNOS– / – mice compared with their age-matched wild type controls (#P=0.03 between eNOS– / – and nNOS+/+ myocytes from older mice, n=25–26).

 
Heart weight and body weight did not differ significantly between eNOS– / – and eNOS+/+ mice at either age (e.g., heart weight was 196±10 mg in young eNOS+/+ mice vs. 187±7 mg in young eNOS– / – mice, P=0.47 and 196±10 mg in old eNOS+/+ mice vs. 208±17 mg in old eNOS– / – mice, P=0.53); similarly, there were no significant differences in basal or ISO+CCh contraction between old eNOS– / – and eNOS+/+ mice. However, the response to ISO was significantly reduced in LV myocytes from old eNOS– / – mice compared with their age-matched wild type controls (8.30±0.26% in old eNOS+/+ vs. 6.52±0.53% in old eNOS– / –, P<0.05).

There was a trend for TR50 to be prolonged in myocytes from older mice (ANOVA, P=0.06, Table 3) and the lusitropic response to ISO tended to be smaller in myocytes from old eNOS– / – mice (reduction in TR50 in ms: 9.74±2.22 ms in young eNOS– / – myocytes vs. 5.62±1.20 in old eNOS– / – myocytes, P=0.06). However, there were no significant differences in TR50 between old eNOS– / – and eNOS+/+ myocytes (ANOVA, P=0.28).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
We tested whether constitutive myocardial NO production from either eNOS or nNOS plays a significant role in the regulation of β-adrenergic and muscarinic cholinergic inotropic and lusitropic responses in isolated LV myocytes. The main conclusions from this study are as follows: 1) muscarinic receptor stimulation by CCh causes the same reduction in β-adrenergic inotropy in myocytes from eNOS– / – mice and wild type littermates under a variety of experimental conditions (e.g., at 1 and 3 Hz stimulation frequency, in the presence of high or low levels of β-adrenergic stimulation, in young or old animals); 2) eNOS gene deletion had no effect on basal or β-adrenergic inotropy and relaxation in LV myocytes (again under a wide range of experimental conditions), whereas targeted disruption of the nNOS gene was associated with a larger contraction and a slower relaxation at all stages of the experimental protocol; 3) the inotropic response to β-adrenergic stimulation differed significantly between eNOS+/+ and C57BL/6J myocytes, old vs. young eNOS– / – and eNOS+/+ myocytes, and between myocytes from old eNOS– / – mice and their age-matched wild type siblings indicating that experimental variables such as age and the choice of control animals may have contributed to the inconsistency in the results reported in the literature.

Our findings suggest that constitutive eNOS expression in the murine ventricular myocardium is not essential for the muscarinic-mediated inhibition of β-adrenergic signalling and is unlikely to play a significant role in the regulation of basal and β-adrenergic inotropy and relaxation under our experimental conditions (i.e., in unloaded LV myocytes). In contrast, nNOS appears to be the myocardial constitutive isoform responsible for the NO-mediated autocrine regulation of myocardial contraction and relaxation.

4.1 Role of eNOS in the regulation of myocardial contraction
Investigations of β-adrenergic signalling in eNOS– / – mice have produced inconsistent results. Gyurko et al. [4] and Gödecke et al. [5] found that the LV inotropic response to β-adrenergic stimulation was greater in isolated hearts from eNOS– / – mice or in vivo. However, eNOS gene disruption has generally been reported to have no effect on β-adrenergic responses in isolated LV myocytes [5,9–11]. These findings could be interpreted as indicating that most of the physiological effects of eNOS-derived NO on myocardial contraction may be paracrine and require intact endothelial membranes. However, recent data have demonstrated a reduction in β-adrenergic responses in the presence of a myocardial-specific overexpression of eNOS [22–26], suggesting that increased NO production in the heart may inhibit β-adrenergic inotropy, regardless of the localisation (myocardial or endothelial) of its source. Conversely, effects of constitutive NO release on the cholinergic control of inotropy have been shown at the single cell level, predominantly in the presence of prior adrenergic stimulation and by using non-isoform specific NOS inhibitors [1,27,28]. Further support for the role of NO in the cholinergic control of adrenergically stimulated ICa and inotropy was shown by studies in LV myocytes from eNOS– / – mice, where there was no significant suppression of ISO-stimulated ICa or contraction in response to muscarinic receptor stimulation [9]. Conversely, eNOS overexpression in LV myocytes has been shown to enhance the negative inotropic effects of muscarinic receptor stimulation both in vivo and in cultured neonatal myocytes [23,25]. There are, however, many contradictory data in the literature (e.g., [22,29,30]). More specifically, several investigators have shown that the muscarinic antagonism of β-adrenergically stimulated ICa or inotropy is preserved in eNOS– / – mice [5,10,11], casting some doubts on the importance of this NOS isoform as an ‘obligatory’ [9,28] mediator of the cholinergic effects on ICa and inotropy.

4.2 Can the controversy in the literature be explained by differing experimental conditions?
4.2.1 Age
It has been suggested that some of the disagreement in the published data concerning the role of eNOS in the regulation of myocardial contraction may be due to the use of mice of different age between experiments, as chronic phenotypic adaptations and the development of LV hypertrophy in older eNOS– / – mice may have confounded the results [18]. We and others [19] have been unable to demonstrate a significant difference in heart weight between eNOS+/+ and eNOS– / – mice at either age, although a small increase in LV myocytes' capacitance (an index of cell surface) has been reported in 3–6 months old eNOS– / – mice compared with age-matched C57BL/6J myocytes [10]. Our data show that β-adrenergic and cholinergic responses decreased with age but failed to demonstrate a difference in the CCh-mediated inhibition of β-adrenergic contraction between old eNOS– / – mice and age-matched controls (eNOS+/+). Conversely, the inotropic response to ISO was significantly smaller in old eNOS– / – myocytes than in their age-matched wild type siblings, suggesting that myocardial adaptations to chronic arterial hypertension [31] may lead to a suppressed β-adrenergic reserve in old eNOS– / – mice. However, differing results were reported by Belevych and Harvey [11] and Han et al. [9], despite the fact that they both used similarly aged eNOS– / – mice (Table 1), suggesting that other methodological variables, such as the choice of control mice, may account for the discrepancy.

4.2.2 Choice of control mice
It is generally accepted that wild type littermate controls, whenever available, make the best controls for mutant mice. Over time, genetic drift can occur in isolated breeding colonies (from different mouse vendors or private research facilities) and wild type littermates best represent the background of the mutant animals. Our experiments clearly show that, had we used C57BL/6J mice as controls, we would have concluded that targeted disruption of the eNOS gene increases β-adrenergic inotropy in isolated LV myocytes, as indeed it was reported by Barouch et al. [6] (Table 1). This is of note because other work (e.g., Han et al. [9]) and this study, found no difference in the inotropic and ICa response of eNOS– / – and eNOS+/+ myocytes to ISO, suggesting that some of the findings attributed to endogenous NO production may reflect the choice of control animals. Similar considerations may explain differences in the inotropic response to CCh between Han et al. [9] and us, as the former used a different source of eNOS– / – mice ([7] vs. [19]). Further variability in experimental findings can stem from differences in the strain of mice used in the backcrosses of the knockout founders (e.g., 129Sv, C57BL/6 or C57BL/6x129Sv; note, for instance, that the inotropic responses to ISO and ISO+CCh are greater in nNOS+/+ myocytes than eNOS+/+ myocytes, Figs. 1 and 2Go) and in the number of backcrosses.

4.2.3 Level of β-adrenergic pre-stimulation and pacing frequency
Reduction of the level of β-adrenergic pre-stimulation from 100 nM to 10 nM ISO as in Han et al. [9] did not alter the effect of eNOS disruption on the CCh response, suggesting that the level of β-adrenergic pre-stimulation does not affect the cholinergic inhibition of β-adrenergic contraction.

Kaye et al. [21] showed that increasing the rate of contraction of LV myocytes from 1 to 3 Hz enhanced the release of endogenous NO, which in turn attenuated the myocytes' force frequency response. Adenoviral or transgenic overexpression of eNOS in LV myocytes has been shown to enhance the cholinergic inhibition of β-adrenergic contraction [23,25]; it is therefore conceivable that the increased NO availability in myocytes field stimulated at 3 Hz may have contributed to the findings of Han et al. [9]. We found that the inotropic and lusitropic response to ISO was smaller at 3 Hz; however, this effect was similar in eNOS+/+ and eNOS– / – myocytes. Similarly, there was no difference in the effect of CCh on ISO-stimulated contraction between genotypes and stimulation frequencies, indicating that the increase in NO production that may be elicited by this manoeuvre is not sufficient to enhance the cholinergic inhibition of β-adrenergic inotropy.

4.3 Role of nNOS in the regulation of myocardial contraction
Our findings confirm that basal contraction is increased and relaxation is impaired in LV myocytes from nNOS– / – mice, as observed in vivo [12,32] and consistent with the idea that myocardial nNOS is the source of NO that is chiefly responsible for the autocrine regulation of myocardial function in healthy young mice. This conclusion is further supported by data indicating that acute pharmacological inhibition of nNOS in LV myocytes isolated from nNOS+/+ mice increases contraction, ICa density and SR Ca2+ content [12] and mimics the enhanced inotropic response of nNOS– / – myocytes to 2 nmol/L ISO [13], thereby recapitulating the myocardial phenotype of nNOS– / – mice.

Stimulation of NO production from the coronary endothelium has been shown to increase LV compliance and hasten myocyte relaxation via a cGMP-dependent reduction in myofilament Ca2+ sensitivity [33–35], whereas non-isoform specific NOS inhibition prolongs LV isovolumic relaxation in mice in vivo [4], indicating that constitutive (presumably eNOS-derived) NO production plays an important role in the regulation of LV diastolic function. Surprisingly, however, Gyurko et al. [4] reported that basal LV relaxation was unaffected in isolated hearts from eNOS– / – mice, which also showed an enhanced lusitropic response to ISO. These findings suggest that (at least in the mouse) nNOS may be the source of NO involved in the regulation of myocardial relaxation. In agreement with this hypothesis, we found no difference in TR50 between eNOS– / – and eNOS+/+ myocytes, whereas relaxation was prolonged in nNOS– / – myocytes at all stages of the experimental protocol (Table 3). It has been previously reported that older (6–12 months of age) nNOS– / – mice develop mild LV hypertrophy [6,32] in the absence of arterial hypertension [36], raising the possibility that time-dependent adaptations to nNOS gene disruption may contribute to the myocardial phenotype of nNOS– / – mice. However, our current findings in young (2–4 months) mice indicate that prolonged relaxation in nNOS– / – myocytes precedes the development of LV hypertrophy. Importantly, we have recently reported that a reduction in phospholamban phosphorylation may be responsible for the slower myocardial relaxation and decay of the intracellular Ca2+ transient in nNOS– / – myocytes and demonstrated that this effect is nNOS-specific as it is reproduced by acute nNOS inhibition and absent in eNOS– / – mice [37]. These findings indicate that a slower Ca2+ reuptake in the SR may account for the impaired LV relaxation of nNOS– / – mice.

We have previously reported an increase in the inotropic response to a low concentration (2 nmol/L) of ISO in LV myocytes from nNOS– / – mice or after acute nNOS inhibition with L-VNIO [13]. However, data obtained by Barouch et al. [6] under different experimental conditions (Table 1) have indicated that the response to β-adrenergic stimulation in nNOS– / – LV myocytes may be biphasic; i.e., enhanced at low ISO concentrations (<10 nmol/L) but greatly attenuated at higher concentrations. Here we found that LV myocytes from nNOS– / – mice have a significantly greater shortening in the presence of 100 nmol/L ISO compared with their wild type littermates; however, the increase in sarcomere shortening in response to ISO did not differ significantly between groups, suggesting that potentiation of the β-adrenergic response in nNOS– / – myocytes only occurs at low concentrations (e.g., <10 nmol/L) of ISO [6,13].

As reviewed in detail previously [14,18,38], differences in subcellular localisation (SR vs. caveolar membrane), mode of activation (via phosphorylation and Ca2+ for eNOS and Ca2+ for nNOS) and rate of NO production (in nmol of NO min– 1: 16 for eNOS vs. 96 for nNOS) between eNOS and nNOS may account for their diverse effects in the myocardium. Recent findings indicating that nNOS translocates from the SR to the plasmalemmal membrane (where it binds to caveolin 3) in the failing myocardium [39] may provide us with a useful model for teasing out the importance of NOS subcellular localisation in determining NO-mediated effects in the heart.

4.4 Conclusions
Taken together, these findings indicate that constitutive eNOS expression in the murine LV myocardium is not essential for the muscarinic-mediated inhibition of β-adrenergic signalling and does not appear to play a significant role in the regulation of basal and β-adrenergic myocardial contraction and relaxation. Our data indicate that nNOS is the constitutive isoform responsible for the NO-mediated autocrine regulation of basal myocardial inotropy and relaxation in the mouse. Given that the inotropic and lusitropic response to 100 nM/L ISO did not differ in nNOS– / – myocytes, our findings suggest that nNOS-mediated attenuation of β-adrenergic responses may only occur at low concentrations of ISO (i.e., <10 nM/L).


   Acknowledgement
 
We are grateful to the British Heart Foundation, the Royal Society and the Garfield Weston Trust for their financial support.


   Notes
 
1 These authors have contributed equally to this work. Back

Time for primary review 32 days


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

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H. Wang, M. J. Kohr, C. J. Traynham, D. G. Wheeler, P. M. L. Janssen, and M. T. Ziolo
Neuronal nitric oxide synthase signaling within cardiac myocytes targets phospholamban
Am J Physiol Cell Physiol, June 1, 2008; 294(6): C1566 - C1575.
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