Cardiovascular Research

Cardiovascular Research 1999 43(3):607-620; doi:10.1016/S0008-6363(99)00163-7
© 1999 by European Society of Cardiology

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Copyright © 1999, European Society of Cardiology

Regulation of cardiac β-adrenergic response by nitric oxide

Jean-Luc Balligand^*

Unit of Pharmacology and Therapeutics, Department of Medicine, University of Louvain Medical School, 53, Avenue E. Mounier, B-1200 Brussels, Belgium

^* Corresponding author. Tel.: +32-2-764-5349; fax: +32-2-764-9322 balligand{at}mint.ucl.ac.be

Received 19 March 1999; accepted 19 April 1999

KEYWORDS Nitric oxide; Adrenergic agonists; Acetylcholine; Heart failure; Contractile function; Experimental studies; Heart; Regulatory mechanisms; Pharmacology; Pathophysiology

	1 Introduction

Top 1 Introduction 2 NOS isoforms in... 3 Regulation of the... 4 Modulation of the... 5 Modulation of the... 6 Intracellular mechanisms of... 7 NO-dependent attenuation of... References

 
The force and frequency of myocardial contraction are physiologically regulated by neurotransmitters and hormones. Norepinephrine released by the sympathetic nerves in the heart and epinephrine released into the circulation by adrenal glands increase myocardial contractility by acting on both - and β-adrenergic receptors on heart muscle. Opposed to that is the action of acetylcholine released from parasympathetic nerves which reduces contractility by binding to muscarinic cholinergic receptors. The first demonstration that these two autonomic pathways are regulated by nitric oxide (NO) produced endogenously within cardiac muscle cells [1] both completes the understanding of the molecular mechanism of action of neurotransmitters in the heart and offers potential new therapeutic approaches for the correction of the altered responsiveness of cardiac muscle to autonomic regulation in circumstances such as heart failure.

We will briefly review the molecular pathways leading to the intracellular actions of β-adrenergic and muscarinic cholinergic agonists in the cardiac myocyte, integrating the more recent evidence implicating the NO pathway from the single myocyte to clinical situations.

1.1 β-Adrenergic pathway
β-Adrenergic agonists are well known to increase the force and frequency of contraction and increase the rate of relaxation of cardiac muscle. Despite some differences between regions of the heart, in general, the early increase in force of contraction is related directly to an increase in transmembrane flux of calcium, through phosphorylation of the L-type Ca channel, and the subsequent calcium-induced calcium release from the sarcoplasmic reticulum, whereas the sustained inotropic effect during the subsequent beats results from both increased influx and increased mobilization of internal calcium resulting from an increased replenishing of the reservoir of calcium in the sarcoplasmic reticulum [2,3].

A hallmark of the β-adrenergic effect on the heart is to increase the rate of relaxation which has been related to more rapid termination of calcium influx, increased rate of calcium uptake and sequestration into the sarcoplasmic reticulum following phospholamban phosphorylation [4,5], increased expulsion of intracellular calcium via sodium/calcium exchange [6], and dissociation of calcium from troponin after the contraction [7,8].

1.1.1 β-Adrenergic receptors
Even though binding studies with selective ligands have revealed two subtypes of β-adrenergic receptors in the heart (β-1 and β-2), the β-1 subtype usually predominates in the mammalian heart. In the rat heart, the percentage of β-1 subtype is 85%. The ratio of β-1 and β-2 differs in different regions of the human heart, where the percentage of β-1 is higher in ventricle than in atrium [9]. Recently, evidence was provided for the expression of β3 adrenoreceptors (at the mRNA level) in biopsy specimens from human ventricles. Functionally, activation of this receptor with specific agonists (in the presence of β1 and β2 blockade) produced a unique negatively inotropic effect on the contraction of human myocardium [10].

1.1.2 Cyclic AMP intracellular pathway
β_1–2-adrenergic receptor stimulation increases cyclic AMP synthesis by stimulating the catalytic activity of adenylyl cyclase via a process involving GTP-binding regulatory proteins containing the _s (or stimulatory) subtype. Adenylyl cyclase belongs to a family of enzymes that convert ATP to cyclic AMP which in turn activates several target molecules, primarily cyclic AMP-dependent protein kinases to regulate the activity of downstream proteins. Even though adenylyl cyclase isoforms are widely distributed, only types V and VI have been definitively identified in ventricular heart tissue. Expression of the type VI isoform is most abundant in the fetus, gradually declines with age and reaches its lowest level in mature adults. As all the other isoforms, these two types are also stimulated by G_s but are unaffected by calcium/calmodulin as opposed to the other types [11].

Many isoforms of adenylyl cyclase, including type V and VI are inhibited by G_i-linked receptors. In addition, type VI adenylyl cyclase can be at least as effectively inhibited by elevations in intracellular calcium, providing a mechanism for the cross-talk between calcium- and cyclic AMP-dependent signaling pathways, both important regulators of cardiac contractility.

1.1.3 Cyclic AMP phosphodiesterases
As for other intracellular cyclic nucleotides, the levels of cyclic AMP are tightly regulated by a complex family of different phosphodiesterases widely distributed among various tissues, including the heart. Among the different members of at least seven families, members of the PDE I, II, and III were clearly shown to be expressed in heart tissue. PDE I is regulated by calcium/calmodulin and therefore the regulation of the overall cyclic nucleotide concentration by this isoform in a cell is expected to be complex because of competition for the active calcium-calmodulin cofactor between the phosphodiesterase and other calcium calmodulin-binding proteins, such as protein phosphatases and constitutive nitric oxide synthases (NOSs). PDE II contains a non-catalytic binding site having high specificity for cyclic GMP. When cyclic GMP binds to this site, it increases the affinity of the catalytic site by allosteric interaction. PDE II is therefore known as a cyclic GMP-stimulated phosphodiesterase. In general, PDE II isoforms have been found to be expressed in tissues in which the effect of cyclic GMP are opposite to those of cyclic AMP. The expression of an isoform of PDE II in heart myocytes from several species, including rat [12,13] is an illustration of this principle. By contrast, PDE III isoforms are inhibited by cyclic GMP. In the heart, such inhibition by cyclic GMP would initially potentiate the increase in intracellular cyclic AMP, thereby producing a positive inotropic effect probably through an increase in L-type calcium current. The net result of cyclic GMP increases in heart muscle cells, however, is more difficult to predict given the co-expression of both PDE II and PDE III isoforms in the same myocytes at least in the rat species [12]. Factors probably concurring to produce either a potentiation or an attenuation of the cAMP effects in heart muscle cells include the relative differences in affinity and V_max for this cyclic nucleotide between the two isoforms, and perhaps the localization of each isoform of PDE together with effector proteins for cAMP in the same subcellular compartment.

1.2 Muscarinic cholinergic pathway
Molecular cloning has allowed the identification of five muscarinic receptor subtypes named m1–m5 based on the order of their discovery [14]. Even though the study of the functional coupling of heterologously expressed receptors has led to the general distinction between those isoforms that either mobilize intracellular calcium (m1, m3 and m5) or inhibit adenylyl cyclase (m2 and m4), this classification is somewhat arbitrary. There is ample evidence that m2 and m4 receptors also do stimulate phospholipase C through a pertussis toxin-sensitive G protein, thereby generating intracellular diacylglycerol (DAG) and IP3 through hydrolysis of membrane phospholipids, with subsequent liberation of calcium from the endoplasmic reticulum [15]. In addition, G protein β subunits were also shown to couple m2 receptor stimulation to phospholipase C β2 [16].

1.2.1 Intracellular effectors for the action of acetylcholine in the heart
Most of the functional effects of acetylcholine in the heart have been attributed to the activation of three main intracellular effectors: activation of potassium channels, inhibition of adenylyl cyclase, activation of soluble guanylyl cyclase leading to increases in intracellular cyclic GMP with subsequent activation of downstream cyclic GMP-dependent effectors. At the outset, one should bear in mind that even though the density of cholinergic innervation differs among regions of the heart with the sinoatrial node and atrioventricular conducting systems being classically under the influence of dense parasympathetic innervation in mammalian hearts, recent work using viral tracing experiments also identified a rich distribution of vagal innervation throughout ventricular cavities [17]. In the absence of β-adrenergic stimulation, however, the effects of acetylcholine are small in ventricular tissue. When the heart is first stimulated by β-adrenergic agonists, the effect of acetylcholine is greatly potentiated, a phenomenon previously termed ‘accentuated antagonism’ [18].

1.2.1.1 I_K-Ach
The activation by acetylcholine of a unique species of potassium channels, designated as I_K-Ach mostly accounts for this phenomenon at the sinoatrial node and atrium, even though this current is also affected by acetylcholine in the absence of catecholamine stimulation. The anatomical distribution of this channel varies greatly in the heart, with very little representation at the ventricular level. In addition the proportion between heart chambers varies according to the species considered with a ventricular I_K-Ach density at about 25% that of the atrium in the frog [19]. In other species, where the channel is present at very low levels in the ventricule, the action of acetylcholine must be accounted for by the activation of alternative pathways.

1.2.1.2 Inhibition of adenylyl cyclase
Among these, the attenuation of adenylyl cyclase activity by muscarinic cholinergic receptors has been well documented in the heart [20]. The sensitivity of this effect to pertussis toxin indicates its mediation through G_i/o proteins. This leads to a decrease in the generation of intracellular cyclic AMP and its downstream positive inotropic effects. Experiments in isolated myocytes from homozygous mice deficient in G-o identified this as the critical isoform for muscarinic receptor coupling to the attenuation of I_Ca-L, at least in the mouse heart [21].

1.2.1.3 NO and cyclic GMP
However, the observation of a dissociation between the functional effects of acetylcholine and cyclic AMP levels [22–24] prompted the search for other second messenger pathways linked to muscarinic receptor stimulation. These include activation of guanylyl cyclase to produce increases in intracellular cyclic GMP. Although a critical review of the early controversial evidence for the role of cyclic GMP as regulator of cardiac contractility is beyond our scope, more recent experiments using exogenous NO donors or cyclic GMP analogues have now established the importance of this cyclic nucleotide in the control of cardiac contractility. As will be developed later on, most of the initial controversy may have stemmed from now well-recognized variations in the observed effects among different regions of the heart, cardiac preparations and animal species used or the concentration of the NO donor or cyclic GMP applied (Fig. 1).

View larger version (41K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Intracellular pathways for the modulation by NO of adrenergic and muscarinic cholinergic signaling. Left: β-adrenergic receptor (β-Adr) stimulation produces a positive inotropic effect through activation of adenylyl cyclase (AC) leading to increases in intracellular cyclic AMP levels. Subsequent activation of cAMP-dependent protein kinase (PKA) leads to downstream phosphorylation of target proteins, including the alpha subunit of L-type calcium channel (bottom) to increase transsarcolemmal calcium influx, phospholamban to increase sarcoplasmic reticulum calcium uptake (middle) or troponin I to desensitize contractile myofilaments to calcium (top). eNOS activation in cardiac myocytes occurs in response to both β-adrenergic and muscarinic cholinergic receptor stimulation. Right: NO activates the soluble isoform of guanylyl cyclase to increase intracellular levels of cGMP, which opposes the positive inotropic effects of the β-adrenergic pathway through (1) activation of the cGMP-stimulated phosphodiesterase (PDE 2) to enhance the breakdown of cAMP (conversely, cGMP can potentiate the effects of cAMP through inhibition of PDE 3); (2) activation of the cGMP-dependent protein kinase (PKG), leading to downregulation of the L-type calcium current (bottom). As for PKA, PKG can decrease myofilament sensitivity to calcium, thereby promoting relaxation, although both pathways may not be operating concurrently. Finally, PKG may enhance sarcoplasmic reticulum calcium release through the cyclic ADP ribose pathway (see text). Light and dark grey arrows denote pathways leading to increase or decrease of excitation–contraction coupling, respectively. In addition, NO produced by eNOS (or iNOS, not shown) may regulate excitation–contraction coupling through mechanisms independent of cGMP elevations (not shown, see text). Aside from eNOS activation, muscarinic cholinergic receptor stimulation results in inhibition of adenylyl cyclase and activation of distinct K+ channels, all of which variably participate in the parasympathetic inhibition of β-adrenergic signaling depending on the species and region of the heart.

 
Despite clear evidence that acetylcholine increases cyclic GMP levels in heart tissue, the coupling of any muscarinic cholinergic receptor to activation of soluble guanylyl cyclase in ventricular myocytes remained poorly understood. Interestingly, Goldberg and Haddox had suggested very early that the intracellular levels of calcium could regulate guanylyl cyclase activation [25]. Combined with the notion that muscarinic cholinergic receptors are clearly coupled to IP3-mediated increases in intracellular calcium as reviewed above, our demonstration of the constitutive expression of a calcium-sensitive isoform of NOS within human and rat ventricular myocytes supported the hypothesis that the parasympathetic regulation of cyclic GMP content in the heart is mediated by NOS [1,26].

In the following sections, we will briefly review the data characterizing the identity of the isoforms of NO synthase expressed in heart muscle and supporting a functional role of NO produced endogenously by NOS in regulating the contractile response to β-adrenergic and muscarinic cholinergic stimulation in whole heart and ventricular myocytes.

	2 NOS isoforms in heart muscle

Top 1 Introduction 2 NOS isoforms in... 3 Regulation of the... 4 Modulation of the... 5 Modulation of the... 6 Intracellular mechanisms of... 7 NO-dependent attenuation of... References

 
There is now ample evidence that all three isoforms of NOS (nNOS, iNOS, and eNOS each encoded by a different gene, NOS1, NOS2 and NOS3, respectively) are expressed within various cell types in the myocardium. In human hearts, eNOS expression has been demonstrated in endothelial cells and cardiac myocytes from atrial and subendocardial ventricular tissue, whereas iNOS immunostaining has been reported, albeit with some variability between studies, in cardiomyocytes, vascular smooth muscle and endothelial cells and infiltrating macrophages (see below).

2.1 nNOS
nNOS has been identified in both cholinergic and non-adrenergic, non-cholinergic nerve terminals, in specialized conduction tissue and in sympathetic nerve terminals of the guinea pig heart [27,28], where it could be immunohistochemically co-localized with tyrosine hydroxylase [29]. Cardiac myocytes, however, do not seem to express the canonical nNOS, at least in the rat [26,30], or the muscle specific isoform of nNOS (nNOS-µ; [31,32]). In rabbit hearts, an NOS isoform with nNOS immunoreactivity but slightly higher molecular weight (M_r 160 000) was recently identified in SR vesicles [33].

2.2 eNOS
Endothelial cells from the endocardium and from arterial capillaries and veins express eNOS in a variety of species, including man [34–37]. In addition, there is unequivocal evidence that eNOS is expressed in cardiomyocytes from atria, atrio-ventricular nodal and ventricular tissue in most mammalian species [26,38,39] and in man [35,36,40]. As in endothelial cells, palmitoylated and myristoylated eNOS in cardiac myocytes is localized to detergent-insoluble glycosphingolipid-rich mycrodomains of the plasmalemma called caveolae [41]. The presence of specific caveolin-binding domains within eNOS allows direct protein protein interaction between the enzyme and the respective cell-specific caveolin isoform, i.e. caveolin 3 in cardiomyocytes and caveolin 1 in endothelial cells [42,43]. Of interest, the recent demonstration that caveolin 3 is expressed in the T-tubular system in cardiac muscle, which had been suggested for years to develop from the coalescence of clusters of caveolae at the sarcolemmal membrane [44] adds weight to the suggestion of a potential role of eNOS in the regulation of excitation–contraction coupling in the heart.

2.3 iNOS
Virtually all cell types within the myocardium can express iNOS upon appropriate stimulation with specific combinations of inflammatory cytokines. These include cardiac microvascular endothelial cells, cardiac myocytes, neurons, vascular smooth muscle cells, and infiltrating inflammatory cells (for a review, see [37]). The relative abundance of iNOS expression within each cell type in vivo may be substantially different from the results observed after exposure of cultured cells to recombinant cytokines in vitro. Of note, some studies have emphasized the relative difficulty of inducing iNOS expression in human cells and the need to use a combination of multiple cytokines, at least in vitro [45]. In addition, immunostaining for iNOS in the myocardium is not only spatially heterogenous but also discontinuous over time, which may explain some of the discrepancy between published reports, especially in human tissue. Finally, the relative expression of iNOS in parenchymal cells versus infiltrating monocytes varies significantly according to the etiology of the cardiomyopathy, e.g. between septic, transplant rejection, ischemic or dilated cardiomyopathies. In human dilated cardiomyopathic hearts, iNOS protein has been colocalized with tumor necrosis factor (TNF), which along with other inflammatory mediators (i.e., IL1, IL6, IFN) was recognized as a component of the ‘innate’ immune response. The fact that the circulating levels of all these inflammatory cytokines were found to be elevated in heart failure raises the question of the role of iNOS expression as an adaptive or maladaptive immune response in heart failure and nevertheless highlights the multiple physiological roles of this isoform of NOS beside the regulation of autonomic stimulation of cardiac contraction.

	3 Regulation of the adrenergic pathway by nNOS

Top 1 Introduction 2 NOS isoforms in... 3 Regulation of the... 4 Modulation of the... 5 Modulation of the... 6 Intracellular mechanisms of... 7 NO-dependent attenuation of... References

 
Several lines of evidence indicate that NO produced endogenously by nerve terminals plays a role in the control of catecholamine release during electrical sympathetic nerve stimulation. This was confirmed in isolated perfused hearts [29] but also in vitro in PC-12 cells [46] and in co-cultures between adult peripheral cardiac neurons and cardiomyocytes [47]. In the latter experiments both exogenous NO release from SNAP and endogenous NO produced by co-cultured intrinsic cardiac neurons acted to increase the spontaneous beating frequency of co-cultured myocytes, whereas NO had no effect on monocultures of cardiomyocytes. These results are consistent with the interpretation that NO either increases the release or inhibits the reuptake of neuron-derived catecholamines at the pre-synaptic level. This interpretation was supported by independent experiments showing that S-nitrosothiols inhibit neuronal norepinephrine transport [46].

The physiological role of nNOS was more directly examined in vivo in homozygous mice deficient for NOS1 (nNOS–/– mice). Under baseline conditions, nNOS–/– mice had a higher mean heart rate and lower heart rate variance than wild-type mice. Their chronotropic response to atropine was significantly blunted, suggesting a reduced baseline parasympathetic tone. However, the bradycardic response to a pressure challenge (phenylephrine injection after β-adrenergic blockade) in nNOS–/– mice was similar to that observed in wild-type controls. The response to pressure challenge was strikingly more sensitive to PTX pre-treatment in nNOS–/– mice compared to wild-type littermate, suggesting that the cardiac inhibitory G_i protein acts in parallel to neuronally-derived NO to mediate autonomic slowing of heart rate in the mouse [48].

	4 Modulation of the β-adrenergic response by eNOS

Top 1 Introduction 2 NOS isoforms in... 3 Regulation of the... 4 Modulation of the... 5 Modulation of the... 6 Intracellular mechanisms of... 7 NO-dependent attenuation of... References

 
As for nNOS, the constitutively expressed eNOS is classically activated by calcium/calmodulin binding to its specific target sequence on each NOS monomer which enables electrons to flow from the enzyme reductase to the oxygenase domain [49]. The EC50 for calcium activation of eNOS enzyme activity is 200–400 nM i.e. within the range of intracellular calcium concentrations observed after the binding of agonists known to elevate intracellular calcium concentrations. Among these, β_1–2-adrenergic agonists are well known to increase intracellular calcium concentration in cardiac myocytes through cyclic AMP and PKA-dependent upregulation of calcium current through voltage sensitive calcium channels (see above). In addition, Gauthier et al. attributed the activation of eNOS in human myocardium to adrenoceptors of the β3 family, for which they had previously identified specific mRNAs in extracts of endomyocardial biopsies. Exposure of these ventricular biopsy fragments to norepinephrine in the presence of full 1- and β1- and β2-adrenoceptor blockade induced an increase in the intracellular concentration of cyclic GMP that was abolished by inhibitors of NO synthase. These effects were reproduced with preferential β3-adrenoceptor agonists, that also elicited increases in NO production directly measurable with a porphyrinic electrode [35].

4.1 eNOS as a negative feedback for the β-adrenergic inotropic effect
The functional consequence of endogenous NO production by eNOS on cardiac contraction is complex, with effects on peak contraction, duration of contraction and onset of relaxation, the direction and magnitude of which vary according to the preparation used and the species considered. In addition, some confusion has arisen when results obtained with endogenous NOS inhibitors were directly compared with those of exogenous NO donor drugs. Experiments with the latter have identified a potentiation or a down-regulation of the contractile response to β-adrenergic agonists in isolated hearts or cardiomyocytes, depending mainly on the concentration of NO applied. Most studies evaluating the contractile effect of NO endogenously produced by eNOS do not identify an enhancement but rather an attenuation of the inotropic effect of catecholamines on heart muscle, as reviewed below. Accordingly, we initially showed that inhibition of eNOS potentiates the positive inotropic response to isoproterenol stimulation in isolated rat ventricular myocytes [25]. A similar observation was later reproduced in rat isolated atria [50] and atrial strips from non-failing human hearts [51]. Furthermore, in rat atrial and ventricular cells as well as in human ventricular tissue, exposure to isoproterenol induced an increase in intracellular cyclic GMP that was significantly reduced with NOS inhibitors [35,50,52].

Several subsequent studies have since validated this paradigm in different animal preparations in vivo, where NO produced endogenously by a constitutive NOS in myocytes and/or neighbouring cells blunts the positive inotropic effect of either infused catecholamines [53], or submaximal electrical stimulation of the left stellate ganglia in dogs [54]. In the latter study, intracoronary infusion of the NOS inhibitor, L-NAME also significantly increased plasma norepinephrine concentration. The concept of eNOS-mediated attenuation of catecholamines’ effect received further confirmation from experiments in mice homozygous deficient for eNOS (eNOS–/–) which exhibited an enhanced contractile responsiveness to positive inotropic agents in comparison to wild-type controls. This finding, independently obtained by two different groups [55,56] was not confirmed in a two other studies using isolated ventricular myocytes from these eNOS–/– mice. The absence of potentiation of the effect of isoproterenol is explained by the use of maximal stimulating concentrations of isoproterenol (1 µmol and above) in one [57]. The presence of significant hypertrophy in the hearts from the older (>3 months) eNOS –/– mice in the other study invalidates the conclusions based on the comparison with normal controls [58].

The activation by catecholamines of inverse inotropic effects can be rationalized in terms of the concurrent activation of a family of adrenergic receptors coupled to different intracellular pathways. This interpretation is supported by the demonstration of the coupling of β3-adrenoceptors to NOS activation in human ventricular tissue with a resultant attenuation of contractile force strikingly contrasting with the classical β_1–2 positive inotropic action of catecholamines (although this does not exclude the possibility of NOS activation by the other β-adrenoceptors) [35]. This paradigm somewhat recapitulates in the heart a similar co-activation of contracting and relaxing effects by catecholamines in the vasculature, where the resulting tone is controlled by the balance between direct vasoconstriction of the smooth muscle and endothelium-derived relaxing factors, including NO. In the heart, the presence of both pathways (positively and NO-dependent negatively inotropic) within the same cardiomyocyte yields a similar negative feedback mechanism but in an autocrine fashion. Predictably, qualitative or quantitative alterations in either limb would alter the responsiveness to adrenergic stimulation and potentially contribute to the development of myocardial dysfunction, as reviewed below.

4.2 eNOS and arrhythmia
eNOS may also modulate other aspects of β-adrenergic stimulation of the heart. Aside from changes in inotropic parameters, we recently observed that treatment of electrically-paced human atrial strips with the NO synthase inhibitor, L-NMMA enhanced the arrhythmogenic effect of submaximal concentrations (10 nM) of isoproterenol, as manifested by the occurrence of aftercontractions [51]. Two additional independent pieces of evidence support a role for NOS in controlling the threshold for adrenergically-induced ventricular arrhythmia. In open chest dogs with acute coronary artery occlusion, Fei et al. showed that intrapericardial perfusion with L-arginine to increase NOS activity (as reflected by an increased NO effluent in the coronary sinus) protected the myocardium against the occurrence of ventricular fibrillation [59]. More recently, the role of myocyte-specific eNOS was directly assessed in the control of ouabain-induced arrhythmias, in which the occurrence of aftercontractions and I_ti currents was compared between ventricular myocytes from eNOS–/– mice and from wild-type controls. The results showed that ouabain induced more arrhythmic contractions and I_ti currents in eNOS–/– myocytes, and that these were efficiently prevented by the exogenous administration of S-nitrosoacetylcysteine, an NO donor [60]. These experiments clearly pointed to a role for myocyte eNOS in controling the arrhythmic threshold and may explain the higher incidence of ventricular arrhythmias in circumstances where eNOS is downregulated. We previously showed such downregulation after intramyocardial elevation of cyclic AMP with drugs such as milrinone [30]. The notion that eNOS may be downregulated in the failing myocardium (see below) may further explain the arrhythmogenic potential of inotropic drugs in patients with heart failure.

4.3 Lusitropic effect of eNOS
In the absence of pre-treatment with catecholamines, stimulation of endocardial endothelial cells of ferret papillary muscles with substance P was shown to produce a shortening of the duration of contraction by inducing earlier onset of relaxation, with little effect on peak force of contraction [61]. Qualitatively similar results were later obtained with bradykinin or substance P in isolated ejecting guinea pig hearts [62]. In ventricular biopsy samples of human transplanted hearts which contain a mixture of endocardial, microvascular endothelial cells and myocytes, activation of eNOS through β3-adrenoceptor stimulation did not significantly alter the shape of electrically-stimulated contractions, i.e. the shortening of the time to half-relaxation was mainly the result of a marked decrease in developed peak tension, without significant change in diastolic relaxation [35]. However, a recent study of the contractile performance of human transplanted hearts in vivo led to the observation that intracoronary administration of substance P reproduced the pattern of earlier onset of relaxation with marginal decrease in peak systolic performance [63], as previously identified in animal papillary muscle or isolated ejecting hearts, as mentioned above. In addition the interplay between β-adrenergic stimulation and the paracrine effect of substance P was examined by monitoring systolic and diastolic parameters in response to substance P infusion under continuous infusion of dobutamine. Even though the results were mainly interpreted as an accentuation of the lusitropic effect of paracrine NO compared to that obtained with substance P alone, the study also showed a significant attenuation of the positive inotropic effect of dobutamine that recapitulates the paradigm of the counterregulatory effect of eNOS on the response to catecholamines as detailed in the previous sections. The mechanism underlying the potentiation of the lusitropic effect of substance P in the context of pre-stimulation with a β-adrenergic agonist remains elusive. It has been proposed to result from synergistic actions of NO-derived cyclic GMP and β-adrenergic-dependent activation of protein kinase A to desensitize contractile myofilaments to calcium. This interpretation, however, is in contradiction with the previous demonstration, in isolated rat cardiomyocytes, that the cyclic GMP and protein kinase G-mediated desensitization of cardiac myofilaments to calcium is abolished upon concurrent stimulation with the β-adrenergic agonist, isoproterenol [64].

4.4 Effects of exogenous NO donors on β-adrenergic response
Despite important biochemical differences between NO as generated from exogenous NO donor drugs or NO endogenously produced by NOS targeted to specific subcellular compartments, the use of nitrovasodilators or similar drugs has been valuable in understanding the potential intracellular mechanisms for the regulation by NO of cardiac function. As previously observed with the regulation of L-type calcium current in isolated myocytes [65,66], exogenous NO donors or cyclic GMP analogs exerted a biphasic effect on β-adrenergically-induced positive inotropic effect depending on the concentration of the NO donor and the resulting increase in intracellular cyclic GMP. A potentiation of the effect of isoproterenol was shown both in isolated ventricular myocytes and papillary muscles from rats [67] and isolated feline papillary muscles [68]. In the latter study, a biphasic effect on baseline developed tension was observed with either 8-bromo-cyclic GMP or treatment with Zaprinast, an inhibitor of phosphodiesterase type V that increases endogenous cyclic GMP levels. Low concentrations of NO donors (and resulting intracellular cGMP levels) increased developed tension whereas higher concentrations resulted in a negative inotropic effect. Interestingly, the transition from positive to negative inotropic effect was shifted to lower concentrations of NO donors or Zaprinast when the muscles had been pre-stimulated with either muscarinic cholinergic agonists or with the β-adrenergic agonist, isoproterenol. Even though the precise mechanism of the sensitization by previous β-adrenergic stimulation to the negative inotropic effect of cyclic GMP remained undetermined from these experiments, it is tempting to speculate that it may be related to the activation by isoproterenol of an endogenous NOS to further increase endogenous cyclic GMP, as observed in rat ventricular myocytes [52] and human ventricular biopsies [35]. The concentration-dependent shift from positively to negatively inotropic effect of cyclic GMP elevation in the presence of β-adrenergic stimulation may not be observed in all species studied. For example, in a recent study on isolated ejecting guinea-pig hearts, Prendergast et al. exclusively observed positive inotropic influences of either exogenous sodium nitroprusside or intracoronary substance P in the context of β-adrenergic stimulation with dobutamine [69]. This positive inotropic influence was manifested only by a change in the decay of the peak developed pressure in response to long term infusion of dobutamine, with no change in peak response to the catecholamine. Surprisingly, contrary to the previously mentioned observations in transplanted patients using similar protocols [63], substance P had no significant effect on the lusitropic effect of dobutamine, emphasizing the need for caution when extrapolating functional results across species.

4.5 Mediation by eNOS of the ‘accentuated antagonism’
As mentioned above, the isoforms of muscarinic receptors predominantly expressed on cardiomyocytes are mainly of the M2 subtype. M2 receptors mediate the negative action of acetylcholine on heart rate and force of contraction through G_-i-mediated inhibition of adenylyl cyclase or G_q coupling to specific potassium currents (I_KAch). In addition, despite differences among mammalian species and regions of the heart there is substantial evidence for an indirect pathway mediating the muscarinic cholinergic attenuation of adrenergic stimulation (‘accentuated antagonism’) through increases in cyclic GMP, as reviewed above. Since a main intracellular activator of the soluble isoform of guanylyl cyclase in many cell types is NO, we first hypothesized that intracardiac increases in cyclic GMP were mediated through muscarinic cholinergic activation of endogenously expressed eNOS. This hypothesis was initially demonstrated by the abrogation of the chronotropic effect of muscarinic cholinergic agonists on spontaneously beating neonatal rat myocytes by inhibitors of NOS [1].

Subsequent negative studies have emphasized that this coupling to NO production may not be operative in all animals species or even in all regions of the heart regardless of species differences. The pathway seems to be functionally absent in frog atrial myocytes [70], ventricular myocytes from adult guinea pigs [71], or in papillary muscle from failing or non-failing human hearts [72]. In a recent study on the L-type calcium current of human atrial myocytes which is sensitive to application of exogenous NO donors, Vandecasteele et al. found no effect of NO synthase inhibitors on the muscarinic cholinergic regulation of either baseline or adrenergically stimulated calcium current [73]. As in most of these negative studies, however, there was no assessment of NO production from these preparations with an independent technique in the particular experimental conditions used.

This and other limitations may also invalidate the negative conclusions from another recent study by the same group [58]. Their contention that eNOS does not mediate parasympathetic regulation of the heart is based on their observation that the responsiveness to muscarinic cholinergic agonists in tissues from mice genetically deficient for eNOS is unchanged from wild-type animals. Foremost is the fact that their eNOS–/– mice were not matched with the appropriate controls, i.e. (1) homozygous founder mice were not back-crossed into the appropriate strains to obtain homogenous genetic background between eNOS–/– and control eNOS+/+ mice, as is advisable in such comparative studies; (2) eNOS–/– mice were studied between 3 and 6 months of age, when the chronic hypertension characteristic of this genetic model had resulted in a significant myocardial hypertrophy, a phenotype not independently accounted for in their comparison with normal controls [74]. In their multicellular (papillary muscle or atrial) preparations, the persistence of the response to muscarinic cholinergic agonists in tissues from eNOS–/– mice may be explained by the activation of I_K-Ach which is known to be present in atrial and ventricular cells [57] and can be upregulated in response to hypertrophic stimuli [75]. Such confounding effects of K⁺ currents should have been controlled by Cs⁺-containing intracellular and extracellular solutions in the experiments with single ventricular myocytes. However, the latter were all performed at room temperature (19–23°C) and the authors again do not provide independent evidence that eNOS (in control myocytes) is active at such low temperatures, thereby preventing any valid comparison. There are precedents for other misleading conclusions based on experiments performed at temperatures well below 37°C (for example see opposite conclusions regarding the functionality of a cADPR pathway in cardiomyocytes in Refs. [76,77]) and this is likely the case with such a sensitive enzymatic activity as eNOS in cardiac myocytes.

By contrast, the involvement of eNOS in parasympathetic signaling was clearly demonstrated from the observation that the accentuated antagonism is completely lost in another study on isolated ventricular myocytes from transgenic mice deficient for eNOS [57]. Importantly, the loss of parasympathetic regulation of L-type calcium current in these myocytes was paralleled by the inability of acetylcholine to increase intracellular cyclic GMP levels, whereas such increase was obtained in myocytes from control animals (where it was also abrogated by NOS inhibitors under identical experimental conditions). Of note, eNOS–/– mice in this study were appropriately back-crossed into C57 B/6 and 129SvEv backgrounds, and studied at a younger age when they were exempt from significant cardiac hypertrophy. Electrophysiological experiments were also performed at 32.5°C. In a subsequent series of experiments, Feron et al. were able to restore the muscarinic cholinergic effect on both spontaneous beating rate and intracellular cyclic GMP increases by transfecting neonatal myocytes from these eNOS-deficient mice with plasmids encoding the wild-type (but not myristoylation-deficient mutant) enzyme. Moreover, consistent with the paradigm of the mutually exclusive interaction of eNOS with either activating calcium/calmodulin or inhibitory caveolin previously demonstrated in endothelial cells, these authors showed that the eNOS-dependent muscarinic cholinergic coupling was interrupted in these ‘knock-in’ experiments upon co-transfection of the ventricular myocytes with plasmids encoding the myocyte-specific caveolin isoform, i.e. caveolin-3, or introduction into these myocytes of small peptides containing the sequence of the caveolin scaffolding domain involved in the specific protein–protein interaction with eNOS resulting in the inhibition of the enzyme [78].

Moreover, the paradigm of eNOS-mediated accentuated antagonism has received confirmation from a number of in vitro and in vivo experiments. In the rat species, eNOS was shown to be expressed in adult ventricular and atrioventricular myocytes, where it mediates acetylcholine’s attenuation of adrenergically prestimulated contraction and L-type calcium current [26,38,79]. Of interest, endogenous NO was also shown to mediate the indirect effects of adenosine on calcium current in rabbit heart pacemaker cells [80]. In the anesthetized ferret adrenergically blocked with propranolol, NOS inhibitors significantly reduced the bradycardia induced by vagal stimulation, an effect that could be reversed by infusion of an excess of L-arginine [81]. Similarly, in the anesthetized dog, infusion of L-NMMA into the sinus and atrioventricular nodal arteries attenuated the negative chronotropic and dromotropic responses to vagal nerve stimulation in the absence or presence of adrenergic activation [82]. NOS inhibition, however, may not always affect the direct cholinergic actions of infused acetylcholine. In a series of experiments in anesthetized rabbits and in isolated guinea pig atria with preserved vagal innervation, Sears et al. outlined the existence of regulatory roles of exogenous or endogenous NO at various levels of cholinergic neurotransmission [83,84]. In guinea pig isolated atria, inhibition of NOS did not affect the maximal rate response to vagal nerve stimulation in adrenergically pre-treated preparations, but significantly slowed down the time course of the vagal negative chronotopic effect. On the basis of the subsequent observation of an acceleration of the rate response to vagal stimulation in the presence of inhibitors of the hyperpolarization-activated current, or I_f, which is known to be stimulated by NO [85], they proposed that the modulatory role of endogenous NO on the vagal response may integrate both a potentiating effect, presumably mediated through inhibition of I_Ca-L, and an antagonistic effect activating I_f, both of which are superimposed on the direct activation by acetylcholine of I_K-Ach, at least at the atrial level. Moreover, these authors observed that application of exogenous NO donors or 8-bromo-cyclic GMP potentiated the chronotropic effect of vagal nerve stimulation but not of directly applied acetylcholine, thereby implicating an additional role of NO at the presynaptic level, a conclusion in agreement with previous results in the anesthetized dog [82]. The existence of functionally opposed effects of NO on the control of heart rate due to interaction with multiple currents (i.e I_Ca-L, I_f but not I_K-Ach) in the atria probably explains the contradictory results provided by different laboratories, depending on the preparation and protocol used (intact vagal nerve stimulation versus direct application of acetylcholine, presence or absence of initial adrenergic stimulation), and the species used (which may affect each current’s relative contribution to the control of heart rate). In the human species, we recently observed a partial abrogation of the accentuated antagonism by NOS inhibitors in electrically-paced atrial strips exposed to acetylcholine after submaximal stimulation with isoproterenol [51].

In this regard, the contribution of eNOS to the accentuated antagonism relative to other intracellular pathways coupled to muscarinic cholinergic receptors may be more identifiable at the ventricular level, given the absence of expression and/or significant functional role of I_f in ventricular cells. As for other muscarinic cholinergic responses, Gi/o proteins appear to be critical in the coupling of the NO-mediated accentuated antagonism. Pre-treatment of rats with pertussis toxin not only abolished the muscarinic cholinergic accentuated antagonism, which was shown to be NO-mediated but also controlled the abundance of eNOS proteins in the isolated hearts from the same animals [86].

	5 Modulation of the β-adrenergic response by iNOS

Top 1 Introduction 2 NOS isoforms in... 3 Regulation of the... 4 Modulation of the... 5 Modulation of the... 6 Intracellular mechanisms of... 7 NO-dependent attenuation of... References

 
There is extensive evidence for the expression of iNOS protein in the multiple cell types comprising cardiac muscle, including in humans, in a variety of pathological conditions such as sepsis, transplant rejection and in certain cases, heart failure. In these circumstances, the physiological/pathological role of NO extends beyond the fine tuning of cardiac contractile response to autonomic stimulation, as is the case for eNOS. NO produced by the ‘high ouput’ iNOS enzyme has been implicated in many aspects of cardiomyocyte biology such as immune defence against intracellular microorganisms, including viruses, and apoptosis (for a review, see [37,87]). Although some or all of these phenomena may significantly bear on the contractile phenotype at the multicellular level, including of the heart in vivo, we will restrict our review on those effects of NO specifically produced by iNOS on the contractile responsiveness to β-adrenergic stimulation.

5.1 iNOS and the β-adrenergic inotropic effect
In the early 1990s, experiments in neonatal cardiac myocytes had shown that cytokine treatment resulted in decreased contractile responsiveness to adrenergic agonists which was associated with an attenuation of the normal increase in intracellular cyclic AMP [88,89]. Using specific combinations of recombinant cytokines or a mixture of inflammatory cytokines obtained from the culture supernatants of rat macrophages stimulated with LPS, we found that the reduced contractile response of isolated ventricular myocytes exposed to submaximal concentrations of isoproterenol was fully reversed upon co-treatment of the cells with NOS inhibitors, thereby clearly implicating NO production in the contractile dysfunction, at least under these experimental conditions [90,91]. Similar findings were subsequently observed using cultures of rat ventricular myocytes in co-culture with iNOS-expressing endothelial cells [92], as well as by many other groups using either isolated contracting cardiomyocytes or papillary muscles exposed to LPS alone or in combination with other cytokines ([93,94]; for a review, see [37]).

Among the inflammatory stimuli that result in iNOS induction and myocardial depression, systemic infusion of endotoxin has been widely studied in animal models because of its potential relevance to human sepsis. In a recent study in conscious mice, administration of endotoxin elevated the concentration of tumor necrosis factor (TNF) and interleukin-6 and resulted in the expression of iNOS with subsequent elevation of circulating nitrite within 4 h of injection. This was accompanied by a significant attenuation of the vasopressor effect of noradrenaline that could be restored upon infusion of L-NMMA [95]. In neonatal rat cardiac myocytes in serum-free cultures, endotoxin similarly induced iNOS activity and production of interleukin-6 and interleukin-1 that were all abolished in the presence of dexamethasone. The expression of these inflammatory mediators blunted the β-adrenenoceptor-mediated increase in contraction amplitude, whereas -adrenoceptor responses where unaltered. The specificity of the effect of NO on β- but not 1-adrenergic actions had previously also been observed in other models [96,97]. Other studies on myocardial depression accompanying septic shock reported that NOS inhibition may reverse only part of the contractile dysfunction [98] or showed a decreased cardiac function in the absence of iNOS induction [99]. Of interest, TNF, which was previously shown to exert a minor role in the induction of iNOS in both microvascular endothelial cells and cardiac myocytes in culture [92,100,101] also produced an attenuation of the β-adrenergic responsiveness in isolated myocytes in a NO-independent manner [96,102,103]. The latter effect may have involved a previously characterized direct effect of TNF on the sphingomyelinase pathway to reduce cardiomyocyte contractile function [104]. This points to the multiplicity of signaling pathways leading to contractile dysfunction in response to inflammatory mediators in circumstances such as sepsis and may provide an explanation for the relative inefficiency of NO synthase inhibitors or anti-TNF antibodies to reverse myocardial depression in some studies. Accordingly, in the human heart, despite the demonstration of increases in iNOS proteins or activity in many disease states with myocardial dysfunction, as mentioned above, the use of NOS inhibitors only variably restored contractile function (for a review, see [100]). Although this would apparently argue against a significant functional role for iNOS in the contractile dysfunction, it should be emphasized that the lack of effect of NOS inhibitors may have been explained by the fact that they were administered at late stages of the dysfunction when cardiomyocytes may have sustained irreversible damage. Clearly, this does not rule out a role for iNOS in the early development of the contractile dysfunction, probably in concert with many other inflammatory mediators (the action of which may also be insensitive to NOS inhibition).

Likewise, the intracellular mechanisms responsible for NO-mediated attenuation of β-adrenergic responsiveness are likely to be complex but may, in part, involve the same mechanisms as those of NO endogenously produced by eNOS, as will be reviewed below.

	6 Intracellular mechanisms of action of NO in cardiac muscle cells

Top 1 Introduction 2 NOS isoforms in... 3 Regulation of the... 4 Modulation of the... 5 Modulation of the... 6 Intracellular mechanisms of... 7 NO-dependent attenuation of... References

 
The effects of NO are classically distinguished between those dependent on cyclic GMP formation following the activation of guanylyl cyclase and those that are independent of cyclic GMP. This classification however does not exclude the possibility that NO acts simultaneously through both mechanisms depending on the amount of NO produced, the intracellular versus extracellular source, intracellular compartmentation of NOS and local redox conditions, all of which are likely to impact on NO reactivity with its intracellular targets. These restrictions aside, the use of exogenous NO donor drugs as well as NOS inhibitors in isolated cells has greatly advanced the understanding of the functional interaction between the NO synthase pathway and β-adrenergic signaling, which will only be considered here (Fig. 1).

6.1 Cyclic GMP-dependent mechanisms
6.1.1 Enhancement of β-adrenergic response
Cardiomyocytes from most mammalian species express type 3 isoforms of phosphodiesterases or PDE3, which are allosterically inhibited by cyclic GMP. PDE3 was suggested to mediate NO-induced increases in cyclic AMP and the subsequent activation of L-type calcium current and inotropy in isolated cardiac myocytes from rodents and also human atria [66,105]. Low concentrations of NO donors also induced a moderate positively inotropic effect in adult rat ventricular myocytes [67] and in open chest dog hearts [106] as mentioned above. In feline cardiac myocytes, the same pathway may mediate the rebound increase in L-type calcium current, calcium transient and contraction after the acute removal of acetylcholine [107,108]. Theoretically, intracellular cyclic AMP levels might also be regulated upstream from phosphodieseterases, i.e. through direct regulation of adenylyl cyclase activity or its coupling to β-adrenoceptor through the stimulatory G-protein, _s. Previous studies in lymphocytes have shown that NO can modulate the GTPase activity of the small G protein, p21-Ras [109,110].

NO-dependent increases in cyclic GMP may also potentiate cardiac contraction through mechanisms independent of cyclic AMP. Accordingly, Galione et al. provided evidence that intracellular cyclic GMP could activate calcium release from the ryanodine channel through activation of ADP ribosyl cyclase and subsequent increases in cyclic ADP ribose [111]. Recently, these authors demonstrated that the pathway may be operative to increase SR calcium release in intact guinea pig cardiomyocytes [77].

6.1.2 Decrease in β-adrenergic response
On the other hand, cardiac myocytes from a variety of species also express the cyclic GMP-activated phosphodiesterase, or PDE2. A rat isoform of PDE2 was recently identified as a main cyclic nucleotide phosphodiesterase isoform in sino-atrial node cells from rabbits [112], where the muscarinic cholinergic ‘accentuated antagonism’ on L-type calcium current was also completely abolished by a specific PDE2 inhibitor, EHNA. A similar pathway was found to be operative in atrio-ventricular and ventricular myocytes from rabbits and rats [26,38,112].

Combined with the notion that cardiomyocytes may express both PDE2 and PDE3, the coexistence of these functionally opposed pathways provides a explanation for the bi-directional effect of NO or cyclic GMP in response to β-adrenergic stimulation of L-type calcium current and contraction [65,67], as mentioned above. In circumstances where high levels of intracellular cyclic GMP are produced, such as those generated upon NO production by iNOS, the resultant activation of PDE2 leads to an attenuation of isoproterenol-stimulated increase in cyclic AMP as well as the shortening of adult rat myocytes in culture [52,90]. A similar mechanism may be operative for the attenuation of β-adrenergic responsiveness by NO produced endogenously by eNOS through its ability to increase intracellular cyclic GMP following adrenergic stimulation, as demonstrated in rat and human ventricular tissue [35,52]. The latter phenomenon could provide an explanation for the fact that NOS inhibitors only affect myocyte contractile shortening when they have been pre-stimulated with β-adrenoceptor agonists [1,90].

In addition to activating PDE2, cyclic GMP can attenuate cardiomyocyte contraction by activating cyclic GMP-dependent protein kinase (PKG). This protein may in turn decrease the L-type calcium current stimulated by cyclic AMP-dependent PKA [113,114], or downregulate the contractile responses of myofilament proteins independently of changes in calcium transients [64,93,115] at least in the absence of β-adrenergic pre-stimulation. Using the model of in vivo endotoxin treatment, Sulakhe et al. studied in parallel the contractile responses to isoprenaline in isolated papillary strips and a variety of intracellular target proteins in cardiomyocytes isolated from endotoxin-injected rats. Attenuated contraction in response to isoprenaline in isolated muscles was paralleled by increased iNOS activity in the myocytes, while the phosphorylations of phospholamban and troponin-inhibitory subunit were decreased compared to extracts from control rats [116]. These results would argue against an effect of iNOS-derived NO on PKG-mediated phosphorylation of troponin I to desensitize cardiac myofilaments to calcium [93].

6.2 Cyclic GMP-independent mechanisms
Accumulating evidence indicates that the oxidation of critical thiol residues on regulatory proteins by NO or peroxynitrite may account for the effects of NO on various parameters of cardiac contraction. These effects are more likely to be produced with high concentrations of NO and superoxide anions in the context of inflammatory cytokine stimulation leading to both the induction of the high-output iNOS and oxidative stress. Among the contractile regulatory proteins sensitive to oxidation, the cardiac calcium release channel (ryanodine receptor) and the L-type calcium channel were shown to be regulated in a bimodal way upon progressive poly-S-nitrosylation [117–120]. NO, and/or peroxynitrite may also potentially regulate key enzymes regulating oxygen consumption and ATP generation within heart muscle, including creatine kinase and cytochrome C oxidase (for reviews see [37,121]). These observations emphasize the need to integrate the various bi-directional effects of NO on excitation–contraction coupling and the need to systematically verify that any effect produced with pharmacological concentrations of NO donors is representative of the action of NO as produced by each specific NOS isoform targeted to sub-cellular compartments.

	7 NO-dependent attenuation of β-adrenergic pathway in heart failure

Top 1 Introduction 2 NOS isoforms in... 3 Regulation of the... 4 Modulation of the... 5 Modulation of the... 6 Intracellular mechanisms of... 7 NO-dependent attenuation of... References

 
Numerous studies have examined the involvement of either eNOS or iNOS in the depressed myocardial responsiveness to catecholamines in heart dysfunction from different etiologies, including sepsis, transplant rejection, ischemic or dilated cardiomyopathies.

All of these pathologies have been shown to be accompanied by increased expression of iNOS in the myocardium, albeit with considerable variability regarding the abundance of iNOS protein and the predominant cellular source as mentioned above. By contrast, although changes in the abundance of eNOS mRNA or protein may not be unidirectional in heart failure of all etiologies, it was shown to be reduced in end-stage failing hearts in ischemic and dilated cardiomyopathies [122] consistent with recent findings at the protein level [123].

When the response to increasing concentrations of isoprenaline was examined in isolated, electrically-stimulated human ventricular myocytes in vitro, the response to β-adrenoceptor stimulation normalized to the maximum shortening induced by high calcium was depressed in myocytes from failing heart compared to those from non-failing hearts. In this model however, co-treatment with the NOS inhibitor L-NMMA did not increase the isoprenaline/calcium ratio in myocytes from failing hearts, under the particular experimental conditions of this study [124]. This contrasts with the results of another study on the isometric contractions of human left ventricular trabeculae. In the latter, muscle strips exhibited a decreased responsiveness to β-adrenergic stimulation manifested by a depressed peak tension and abbreviated early relaxation time. These alterations were significantly correlated with the abundance of iNOS activity and mRNA in the same hearts. Importantly, these alterations were corrected upon treatment of the muscles with the NO synthase inhibitor, L-NMMA [122]. As mentioned previously, the inotropic effect of dobutamine was similarly attenuated upon intracoronary infusion of substance P (expected to increase paracrine production of NO) both in non-failing transplant recipients and in patients with dilated non-ischemic cardiomyopathy. In the latter study, this cardiodepressant effect was accompanied with a potentiation of the lusitropic effect of substance P [63]. Furthermore, Hare and colleagues observed a potentiation of the inotropic response to peripheral infusion of dobutamine after intracoronary administration of L-NMMA in patients with dilated cardiomyopathy [125,126]. The contrast with unaltered responsiveness to the catecholamine in normal patients was interpreted as an increased sensitivity of the failing heart to NOS inhibition, at least in vivo. These observations would appear to validate other studies on cardiomyocytes isolated form animal models of heart failure [127]. Finally, the ability of exogenous NO donors to produce quantitatively and qualitatively similar effects in isolated atrial and ventricular strips from human failing and non-failing hearts adds further evidence for a significant role of NO as a modulator of β-adrenergic responsiveness [128].

Time for primary review 12 days.

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Top 1 Introduction 2 NOS isoforms in... 3 Regulation of the... 4 Modulation of the... 5 Modulation of the... 6 Intracellular mechanisms of... 7 NO-dependent attenuation of... References

 

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