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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1846-1858.)
© 1997 American Heart Association, Inc.

Articles

Nitric Oxide Synthases and Cardiac Muscle

Autocrine and Paracrine Influences

Jean-Luc Balligand; ; Paul J. Cannon

From the Department of Medicine, Pharmacology Unit, FATH 53.49, University of Louvain Medical School, Brussels, Belgium (J.-L.B.), and the Department of Medicine, Division of Cardiology, Columbia University College of Physicians and Surgeons, New York, NY (P.J.C.)

Correspondence to Paul J. Cannon, the Department of Medicine, Division of Cardiology, Columbia University College of Physicians and Surgeons, 630 W 168th St, New York, NY 10032. E-mail pjc4{at}columbia.edu

	Abstract

Top Abstract Introduction NOS nNOS iNOS eNOS Molecular Targets of NO Biological Effects of... Biological Effects of Myocardial... Biological Roles of NO... Mechanisms by Which NO... References

 
Abstract The different cell types comprising cardiac muscle express one or more of the three isoforms (neuronal NOS, or nNOS; inducible NOS, or iNOS; and endothelial NOS, or eNOS) of nitric oxide synthase (NOS). nNOS is expressed in orthosympathetic nerve terminals and regulates the release of catecholamines in the heart. eNOS constitutively expressed in endothelial cells inhibits contractile tone and the proliferation of underlying vascular smooth muscle cells, inhibits platelet aggregation and monocyte adhesion, promotes diastolic relaxation, and decreases O₂ consumption in cardiac muscle through paracrinally produced NO. eNOS is also constitutively expressed in cardiac myocytes from rodent and human species, where it autocrinally opposes the inotropic action of catecholamines after muscarinic cholinergic and ß-adrenergic receptor stimulation. iNOS gene transcription and protein expression are induced in all cell types after exposure to a variety of inflammatory cytokines. Aside from participating in the immune defense against intracellular microorganisms and viruses, the large amounts of NO produced autocrinally or paracrinally mediate the vasoplegia and myocardial depression characteristic of systemic immune stimulation and promote cell death through apoptosis. In cardiac myocytes, NO may regulate L-type calcium current and contraction through activation of cGMP-dependent protein kinase and cGMP-modulated phosphodiesterases. Other mechanisms independent of cGMP elevations may operate through interaction of NO with heme proteins, non-heme iron, or free thiol residues on target signaling proteins, enzymes, or ion channels. Given the multiplicity of NOS isoforms expressed in cardiac muscle and of the potential molecular targets for the NO produced, tight molecular regulation of NOS expression and activity at the transcriptional and posttranscriptional level appear to be needed to coordinate the many roles of NO in heart function in health and disease.

Key Words: nitric oxide synthases • cardiac muscle • cytokines • heart failure

	Introduction

Top Abstract Introduction NOS nNOS iNOS eNOS Molecular Targets of NO Biological Effects of... Biological Effects of Myocardial... Biological Roles of NO... Mechanisms by Which NO... References

 
The nomination of nitric oxide (NO) as Science's "Molecule of the Year 1992" was a consecration for a seemingly common gas otherwise known as a component of cigarette smoke and an atmospheric pollutant. It was justified, however, by the discovery of the key signaling roles of NO in as diverse physiological functions as neurotransmission in the peripheral and central nervous systems, immune defense against microorganisms, and modulation of vascular tone.^{1 2 3 4}

Aside from the well defined role of NO (and its derivatives) as an endothelium-derived relaxant of underlying vascular smooth muscle, much interest has recently been focused on the role of endogenous NO pathways as paracrine or autocrine regulators of cardiac muscle function. After a brief description of the biochemistry and molecular regulation of the three isoforms of nitric oxide synthase (NOS) and the general features of NO reactivity relevant to its physiological effects, we review the evidence on the role of NO produced by endothelial NOS (eNOS) or inducible NOS (iNOS) in regulating cardiac muscle function. We end with a brief review of the mechanisms by which NO regulates cardiac cell contraction.

	NOS

Top Abstract Introduction NOS nNOS iNOS eNOS Molecular Targets of NO Biological Effects of... Biological Effects of Myocardial... Biological Roles of NO... Mechanisms by Which NO... References

 
The three NOS isoforms originally identified in the brain (neuronal NOS, or nNOS),⁵ macrophages (inducible NOS, or iNOS),^{6 7} and endothelial cells (endothelial NOS, or eNOS)^{8 9 10} share 50% to 60% homology of their amino acid sequence and are encoded by three different genes (designated NOS 1, NOS 2, and NOS 3, respectively). All three isoforms combine two functionally complementary portions, a carboxyl-terminal reductase domain homologous to cytochrome P450 reductase and an amino-terminal oxygenase domain containing binding sites for heme, L-arginine, and tetrahydrobiopterin (THB4), the two portions being connected by a calmodulin-binding domain in the middle. In addition, on activation, the three isoforms presumably function as homodimers. Within each monomer, electrons provided by NADPH are transferred from the flavins (FAD or FMN) in the carboxyl-terminal portion of the molecule to heme iron, which is activated to bind O₂ and, in the presence of the substrate L-arginine, to catalyze the synthesis of NO and L-citrulline. The binding of calmodulin appears to be required to enable electron transfer from the flavin prosthetic groups to heme.^{11 12 13} In nNOS and eNOS, physiological concentrations of calcium regulate calmodulin binding to its specific domain and the flow of electrons to heme; in iNOS, calmodulin is tightly bound even at low concentrations of intracellular calcium.^{11 12} In addition to the binding of calmodulin, activation of all three NOSs requires THB4, which appears to enable electron transport by stabilizing the homodimeric conformation of the enzymes.^{14 15 16 17} Subunit dimerization of nNOS and the binding of THB4 also appear to be dependent on the presence of the heme prosthetic group.¹⁸ During NO synthesis, L-arginine is first hydroxylated to the intermediate N-hydroxyl-L-arginine, which then undergoes further oxidation to yield NO and L-citrulline. If the substrate L-arginine is unavailable in sufficient amounts, the electron transfer after oxidation of NADPH in nNOS and eNOS is to oxygen, leading to formation of superoxide anions and H₂O₂.^{19 20 21 22} In the nNOS and iNOS isoforms, there is evidence that NO may exert negative feedback on NO synthesis by forming ferrous nitrosyl complexes in their heme prosthetic groups, which partially self-inactivate the enzymes.^{23 24 25 26} In activated macrophages, NO can inhibit iNOS activity by reducing heme availability and its insertion into monomers, thus blocking the dimerization required for enzyme activity.²⁷ A 10-kDa protein that interacts with nNOS and inhibits its activity, designated PIN, has recently been identified; it binds to nNOS and destabilizes the dimer conformation necessary for activity.²⁸

	nNOS

Top Abstract Introduction NOS nNOS iNOS eNOS Molecular Targets of NO Biological Effects of... Biological Effects of Myocardial... Biological Roles of NO... Mechanisms by Which NO... References

 
The gene coding for nNOS, NOS 1, is located on human chromosome 12. Twenty-nine exons encode various alternatively spliced mRNAs that are differentially expressed among various tissues, which are translated into proteins of 160 kDa.^{5 29} The amino acid sequences are highly conserved between species, with 93% identity at the amino acid level between rats and humans. The use of at least eight different exon 1's, each under the control of a distinct 5'-flanking region, contributes to the tissue and developmental stage heterogeneity of generated cDNAs.³⁰ In skeletal muscle, nNOS contains 34 additional amino acids compared with the cerebellar protein, owing to a 102-bp segment alternatively spliced between exons 16 and 17. This isoform, termed nNOSµ, is detected only in differentiated striated muscle.³¹ nNOS protein is expressed in neurons and epithelial cells but not in isolated cardiac myocytes (at least in the rat species). nNOS is expressed in cholinergic and nonadrenergic, noncholinergic nerve terminals, in specialized conduction tissue in the heart^{32 33} , and in sympathetic nerve terminals, where it has been postulated to play a role in catecholamine release and reuptake.^{34 35} nNOS phosphorylation by a variety of protein kinases (calcium/calmodulin–dependent, cAMP-dependent, and cGMP-dependent protein kinases and protein kinase C [PKC]) results in diminished enzyme activity, at least in vitro.^{36 37} Dephosphorylation of nNOS by calcineurin produces the opposite effect.³⁸

	iNOS

Top Abstract Introduction NOS nNOS iNOS eNOS Molecular Targets of NO Biological Effects of... Biological Effects of Myocardial... Biological Roles of NO... Mechanisms by Which NO... References

 
The gene encoding iNOS, NOS 2, is located on human chromosome 17 and contains 26 exons.³⁹ The cDNA encodes a protein of 130 kDa that in the mouse (and partially in humans) tightly binds calmodulin even when intracellular calcium levels are low.^{39 40 41 42} NOS 2 was first cloned and characterized in macrophages,^{6 7} but iNOS activity has been detected in a variety of cell types, including endocardial and endothelial cells, vascular smooth muscle cells, fibroblasts, and neonatal and adult cardiac myocytes from several species.^{43 44 45 46 47 48} iNOS expression is induced by a wide variety of agents, including endotoxin (lipopolysaccharide [LPS]), interleukin (IL)-1ß, tumor necrosis factor (TNF)-, interferon (IFN)-, and IL 6, reflecting the complexity of and differences in the regulatory elements in the NOS 2 promoter in both murine and human species.^{42 49 50} In the 5'-flanking region of the NOS 2 gene in both mice and humans, multiple consensus sequences for the binding of transcription factors that mediate responsiveness to cytokines have been identified, including those for nuclear factor (NF)-B, IFN-, NF-1, IL-6, and interferon regulatory factor (IRF)-1 (which appears to be critical for iNOS expression in murine macrophages).^{42 49 51 52 53} Even within the same rodent species, exposure to the same stimuli results in differential expression of iNOS transcripts among various cell types, reflecting the accessibility of the NOS 2 promoter to a variety of transcription factors generated by cell-specific signaling pathways. For example, IFN- stimulates iNOS mRNA accumulation in cardiac myocytes but not in microvascular endothelial cells from adult rat hearts.^{54 55 56} In both of the latter two cell types, IFN-–induced signal transducer and activator of transcription-1 (STAT-1) phosphorylation was apparent, but induction of iNOS mRNA expression occurred in myocytes only. In the myocytes, induction of iNOS by IFN- was blocked by benzodiazepine peptidomimetic (BZA 5B), an agent that blocked activation of the 44- and 42-kDa mitogen–activated protein kinases (MAPKs; ERK1/ERK2). In microvascular endothelial cells, treatment with the phosphatase inhibitor okadaic acid in the absence of cytokines activated ERK2 and induced accumulation of iNOS transcripts.⁵⁶ These results suggested that activation of ERK1/ERK2 was required for induction of iNOS by cytokines in both cell types and that in microvascular endothelial cells, phosphorylation of STAT-1 is necessary, but is not in itself sufficient for NOS 2 gene expression.

Many factors appear to be involved in the complex regulation of iNOS induction in response to cytokines.^{57 58} Among these, activation of angiotensin II⁵⁹ and -adrenergic receptors,⁶⁰ activation of PKC isoforms,⁶¹ and increases in cAMP^{62 63} all promote iNOS expression in cardiac myocytes, where the effects of cAMP have been shown to involve increases in iNOS mRNA stability.⁶² Conversely, when iNOS has been induced by cytokines in cardiac myocytes, the generation of cAMP in response to ß-adrenergic agonists is blunted in these cells.⁶⁴ Transforming growth factor (TGF)-ß, which stimulates iNOS expression in 3T3 fibroblasts,⁶⁵ diminishes iNOS expression in response to cytokines in microvascular endothelial cells, cardiac myocytes, and vascular smooth muscle cells.^{44 45 54 66 67 68} In the latter cells, inhibition involves regions in the promoter/enhancer region of NOS 2 other than the NF-B site.⁶⁹ Salicylate or aspirin suppress iNOS induction and activity in cardiac fibroblasts in vitro,⁷⁰ and glucocorticoids suppress iNOS induction by cytokines in microvascular endothelial cells and cardiac myocytes, in part by increasing expression of the matrix phosphoprotein osteopontin, which acts to decrease iNOS expression.⁷¹

The availability of cofactors and substrate also influences iNOS expression and NO biosynthesis. Cytokine induction of iNOS in macrophages, microvascular endothelial cells, and cardiac myocytes is accompanied by coinduction of GTP cyclohydrolase, the key enzyme for de novo biosynthesis of THB4 (the induction of which can also be downregulated by dexamethasone).^{54 72} In addition to stabilizing iNOS mRNA,⁷³ THB4 can limit the rate of NO synthesis, presumably through its role, along with heme and L-arginine, in stabilizing the iNOS dimers required for full enzyme activity (see above).⁷⁴ In wounds it has been demonstrated that the concentration of L-arginine can also be rate limiting for NO synthesis by macrophage iNOS.^{75 76} Cytokine treatment of macrophages, microvascular endothelial cells, or cardiac myocytes results in the coinduction of iNOS and the cationic amino acid transporter proteins CAT1, CAT2B (both high affinity), and CAT2A (low affinity), which increase intracellular substrate availability.⁷⁷ When the intracellular level of L-arginine is reduced by competitively inhibiting its transport with the amino acid L-lysine or, in macrophages, by multivalent guanylhydrazone CNI 1493, nitrite release is significantly reduced from these cells.⁷⁸

	eNOS

Top Abstract Introduction NOS nNOS iNOS eNOS Molecular Targets of NO Biological Effects of... Biological Effects of Myocardial... Biological Roles of NO... Mechanisms by Which NO... References

 
The human gene encoding eNOS, NOS 3, is located on human chromosome 7.^{79 80} NOS 3 comprises 26 exons and its cDNA, first characterized in bovine aortic endothelial cells,^{8 9 10} encodes a 135-kDa enzyme protein that is activated by increases in intracellular calcium. eNOS has been shown to be constitutively expressed in endocardial cells; endothelial cells of arteries, veins, and capillaries; kidney epithelial cells; hippocampal pyramidal neurons; skeletal myocytes; and cardiac sinoatrial, atrioventricular nodal, and ventricular myocytes from rodent species.^{4 81 82 83 84 85 86} In addition, there is unequivocal evidence for eNOS expression in atrial and ventricular myocytes from human hearts.^{81 87 88} The promoter region of the NOS 3 gene contains several cis-regulatory sequences, including AP-1, AP-2, NF-1, cAMP, Sp-1, and GATA response elements, the latter two being critical for basal promoter activity in large-vessel endothelial cells.^{89 90} In addition, shear stress–and sterol-responsive elements are probably involved in the upregulation of eNOS in endothelial cells in response to shear stress, exercise, and estradiol.^{9 10 80 91} NOS 3 transcription has also been reported to increase in response to lysophosphatidylcholine and oxidized LDL.^{92 93} In cardiac myocytes from adult rats, treatment with cytokines such as IFN- and IL-1ß is associated with decreased eNOS transcripts by a mechanism that is currently undefined.⁸¹ In endothelial cells from large vessels, TNF- destabilizes eNOS mRNA.⁹⁴ In studies of rat cardiac myocytes in vitro and in vivo, interventions that increased cAMP, such as administration of forskolin, milrinone, or 8-bromo-cAMP, were associated with reductions in eNOS mRNA and protein, which appeared to involve a transcriptional mechanism because eNOS mRNA half-life was unchanged.⁹⁵ This effect of cAMP appeared to be cell type specific, since it did not occur in microvascular endothelial cells from the same hearts.

eNOS undergoes significant posttranslational modifications. In both endothelial cells and cardiomyocytes, the enzyme is mainly associated with membrane fractions, where it is both myristoylated and palmitoylated.^{81 96 97 98} Mutation of the N-myristoylation site converts eNOS from a membrane-associated to a cytosolic protein.^{96 97} Bradykinin promoted the depalmitoylation of eNOS and its translocation to the cytosol in one study⁹⁸ but not in another.⁹⁹ Recombinant eNOS can also be phosphorylated by PKC, PKA, and calmodulin kinase 2.¹⁰⁰ However, these in vitro phosphorylations differ from the phosphorylation pattern induced by bradykinin in intact endothelial cells, which accompanies translocation of the enzyme from the particulate to cytosolic fractions.¹⁰⁰

Recently it was discovered that palmitoylated and myristoylated eNOS in both endothelial cells and cardiac myocytes is localized to caveolae and the detergent-insoluble glycosphingolipid–rich microdomains in the plasmalemma.^{101 102} Caveolae and these microdomains facilitate the transport of molecules across cells and are sites where G proteins and other molecules involved in signal transduction are compartmentalized.^{103 104} Caveolins, the principal structural proteins of caveolae, may cycle between plasma membranes and the Golgi apparatus, where eNOS has also been localized in endothelial cells.^{105 106 107} Recent data indicate that eNOS is coimmunoprecipitated with caveolin 1 in extracts of microvascular endothelial cells and with the myocyte-specific isoform caveolin 3 in extracts of cardiac myocytes.¹⁰⁸ The relationship between the intracellular compartmentalization of eNOS and its enzyme activity in both endothelial and heart muscle cells awaits further study.

NO synthesis by eNOS in endothelial cells is transiently increased by agonists such as bradykinin and substance P, which promote increases in intracellular calcium after interaction with their receptors; bradykinin may also act through increases in intracellular pH.^{109 110} NO synthesis by eNOS in response to shear stress is more sustained, persists independently of calcium transients, and involves tyrosine phosphorylation and interactions with other molecules in caveolae of the plasmalemmma.^{109 111 112} Endothelial cell calcium transients and presumably NO synthesis are also stimulated by other deformations of the cell, such as tapping, stretching, or compression.^{113 114 115} The complex calcium-independent molecular mechanisms responsible for sustained NO production by eNOS in response to shear stress are currently under active investigation in many laboratories. In cardiac myocytes, eNOS is also activated by increases in beating rate^{116 117} and hypoxia.¹¹⁸

	Molecular Targets of NO

Top Abstract Introduction NOS nNOS iNOS eNOS Molecular Targets of NO Biological Effects of... Biological Effects of Myocardial... Biological Roles of NO... Mechanisms by Which NO... References

 
Knowledge concerning the molecular targets of NO continues to expand rapidly (the Table). NO diffuses rapidly and isotropically through cell membranes in most tissues. The concentration of NO within a cell may be equated to the sum of the NO synthesized in that cell plus the NO synthesized by nearby cells that enters by diffusion. Biological effects of NO are likely to be modulated in different microenvironments by (1) the amount of NO produced (eg, synthesis of NO by high-output iNOS tends to be greater and more prolonged than by eNOS), (2) the number and variety of available molecular targets within diffusion distance from the source of NO synthesis, along with their relative reaction rates with NO; and (3) the net redox state that prevails. Among the predominant targets of NO are heme proteins, particularly the heme moiety of soluble guanylyl cyclase, which undergoes conformational change on interaction with NO that leads to enzyme activation and increased synthesis of cGMP.¹¹⁹ This cyclic nucleotide in turn mediates the activation of PKG and other kinases, followed by phosphorylation of several target regulatory proteins, modulation of ion channel function, and physiological responses.^{1 119} NO is trapped and inactivated by interaction with heme moieties in oxyhemoglobin and myoglobin.¹²⁰ The prostaglandin- and thromboxane- forming activity of heme-containing cyclooxygenase 2 is enhanced by NO,^{121 122} whereas the enzymatic activity of heme-containing NOS is inhibited (see above). NO also binds to non–heme iron, particularly iron-sulfur complexes in enzymes such as cis-aconitase, NADH succinate oxidoreductase, NADH ubiquinone oxidoreductase (mitochondrial complexes I and II), and cytochrome c oxidase (complex IV).^{2 123 124} NO can inhibit DNA synthesis by binding the non–heme iron of ribonucleotide reductase.^{125 126}

Other major targets of NO reactivity are reduced thiol residues on most intracellular, membrane-associated, or plasma proteins, such as albumin or tissue plasminogen activator.^{120 127 128} Recently, hemoglobin itself has been shown to be nitrosylated at sites protected from scavenging by heme, which would allow allosterically controlled exchange of NO to other acceptors and export of NO-related vasoactive molecules (presumably S-nitrosothiols) from erythrocytes to regulate vasomotor tone and oxygen delivery to peripheral tissues.¹²⁹ Nitrosylation of other proteins may also affect their function positively or negatively, such as inactivation of the NMDA receptor^{120 127 130} or activation of the calcium-activated potassium channel.¹³¹ NO stimulates the auto–ADP-ribosylation of GAPDH, which in addition to interactions at the active site of the enzyme, may result in inhibition of glycolysis.^{132 133 134} By forming S-nitrosoglutathione, NO can deplete intracellular stores of reduced glutathione, resulting in activation of the hexose monophosphate shunt.¹³⁵ NO can also directly inhibit NADPH oxidase.¹³⁶

NO also interacts with oxygen radicals. Interaction with superoxide leads to formation of peroxynitrite (ONOO^-), which in low concentrations can release NO but at high concentrations is a potent oxidant that can oxidize lipids, directly nitrate tyrosine residues on proteins, and decompose to toxic hydroxyl radicals.^{137 138} Nitration of actin and/or other cytoskeletal proteins in cardiovascular cells by peroxynitrite can alter their structure and may have deleterious effects on the function of contractile myofilaments.^{138 139 140} NO (or a redox-related derivative, such as peroxynitrite) interacts with proteins involved in iron metabolism, liberating iron from cytosolic aconitase¹⁴¹ and allowing interaction of apo-aconitase (or iron response–element binding protein [IRE-BP]) with target sequences in the 5'-untranslated region of ferritin mRNA to inhibit its translation. Binding of the IRE-BP to the 3'-end of transferrin receptor mRNA stabilizes the mRNA and results in increased synthesis of the corresponding protein.^{142 143 144} Peroxynitrite inactivates the mitochondrial aconitase involved in the tricarboxylic acid cycle.¹⁴⁵ NO in large amounts can also promote DNA strand breaks, which can initiate apoptosis or activate poly-ADP ribose synthetase, leading to depletion of cell energy stores and cell death of neurons^{146 147 148} or suppression of contractile activity, at least in vascular smooth muscle.¹⁴⁹

	Biological Effects of Endothelial and Endocardial eNOS

Top Abstract Introduction NOS nNOS iNOS eNOS Molecular Targets of NO Biological Effects of... Biological Effects of Myocardial... Biological Roles of NO... Mechanisms by Which NO... References

 
Paracrine and autocrine influences of NO on various cell types composing cardiac muscle are summarized in Figs 1 and 2. NO inhibits both platelet adhesion to endothelial cells and platelet aggregation (see Fig 1; see also References 150 and 151^{150 151} ). NO also reduces monocyte adhesion to the endothelium and expression of monocyte chemoattractant protein (MCP)-1, which has been implicated in early monocyte recruitment during atherogenesis.^{152 153 154 155} NO reduces both the activation of NF-B and the induction in endothelial cells of the leukocyte (vascular cell) adhesion molecule (VCAM)-1 by cytokines and by oxidized LDL.^{156 157 158 159} NO also inhibits migration¹⁶⁰ and proliferation of vascular smooth muscle cells in vitro.¹⁶¹ In experiments in which eNOS cDNA was transfected into rat carotid arteries, there were significant reductions in neointimal hyperplasia after balloon injury.¹⁶²

View larger version (38K): [in this window] [in a new window]   Figure 1. Paracrine and autocrine influences of NO produced by endothelial NO synthase (eNOS) in cardiac muscle. Endothelial cell eNOS activated by shear stress and various agonists exerts paracrine influences on platelets to inhibit aggregation, on vascular smooth muscle cells to induce relaxation and inhibit proliferation, and on cardiac myocytes to increase diastolic relaxation and decrease O2 consumption. Norepinephrine and acetylcholine released from autonomic nerve terminals stimulate their respective receptors to activate eNOS expressed in cardiac myocytes. The NO produced opposes the positive inotropic effect of norepinephrine (also see Fig 3). Ach indicates acetylcholine; Nad, norepinephrine; BK, bradykinin; CM, cardiac myocyte; EC, endothelial cell; VSMC, vascular smooth muscle cell; PL, platelet; and Subst, substance.

View larger version (37K): [in this window] [in a new window]   Figure 2. Paracrine and autocrine influences of NO produced by inducible NO synthase (iNOS) in cardiac muscle. On exposure to cytokines and other inflammatory mediators, iNOS gene transcription is induced in all cell types and in infiltrating interstitial macrophages. The large quantities of NO produced autocrinally or paracrinally by the high-output enzyme decrease the contraction of cardiac myocytes and vascular smooth muscle cells through various mechanisms (also see Fig 3 and the Table). NO produced by iNOS also induces apoptosis in various cell types, including cardiac myocytes. MP indicates macrophage. See Fig 1 legend for explanation of other abbreviations.

NO synthesized by eNOS in endocardial¹⁶³ and endothelial cells in response to bradykinin and substance P has been shown to have paracrine effects in ferret papillary muscles and in isolated guinea pig hearts; endogenous and exogenous NO accelerated diastolic ventricular relaxation without significant effects on the rate of systolic force development.^{164 165} Similar results were obtained in patients after intracoronary infusions of substance P to stimulate endothelial cell eNOS activity and paracrine NO production.¹⁶⁶ Studies in isolated, perfused rat hearts equipped with a porphyrinic microsensor to measure NO in myocardial tissue indicated that NO release occurred in diastole during each cardiac contraction, was increased by increases in preload, and was stimulated by factors that increased myocardial compression, such as increases in end-diastolic volume or transmural pressure.¹⁶⁷ Because isolated endocardial and endothelial cells can increase NO synthesis in response to external force and because cardiac NO synthesis in response to applied force declined when cardiac endothelial cells were denuded with Triton X-100, the investigators attributed the source of NO synthesis induced by mechanical forces in the hearts to intramyocardial endothelial cells.¹⁶⁷ In settings of increased heart rate and coronary flow (such as exercise), it has been suggested that the lusitropic effect of endothelium-derived NO may benefit both subendocardial coronary perfusion and diastolic ventricular filling.¹⁶⁸ The paracrine effect of endothelium-derived NO on cardiac myocyte function was directly demonstrated in vitro by another group of investigators who used bradykinin to stimulate NO synthesis by endothelial cells in coculture with isolated guinea pig cardiac myocytes (which lack bradykinin receptors) and observed a decrease in contractility of the myocytes in response to bradykinin that was reversed by NOS inhibitors or NO scavengers, such as hemoglobin and methylene blue.¹⁶⁹

Additional evidence for paracrine effects of microvascular endothelial cell NO on adjacent myocardial cells was provided by investigators who measured oxygen consumption of isolated segments of canine myocardium with an oxygen electrode.¹⁷⁰ Incubation of the segments with NO-donor drugs, bradykinin, or the muscarinic agonist carbachol was associated with significant reductions in oxygen consumption of the myocardial segments. The effects of the latter two agonists were abrogated by an inhibitor of NO synthesis. Because the investigators were unable to detect NO formation by isolated canine cardiac myocytes, they suggested that microvascular endothelial NO synthesis may exert a negative influence on mitochondrial respiration (and potentially, energy formation) in adjacent heart muscle cells.¹⁷⁰ In myocardial segments from dogs with pacing-induced heart failure (which had higher than control rates of myocardial oxygen consumption), bradykinin and carbachol did not exert similar depressant effects on myocardial oxygen consumption.¹⁷⁰

	Biological Effects of Myocardial eNOS

Top Abstract Introduction NOS nNOS iNOS eNOS Molecular Targets of NO Biological Effects of... Biological Effects of Myocardial... Biological Roles of NO... Mechanisms by Which NO... References

 
Aside from endocardial and endothelial cell–derived NO, evidence has been provided for a functional effect of NO produced by eNOS expressed in cardiac myocytes themselves (Fig 1). In studies of papillary muscles and of isolated cardiac myocytes from rats, increases in NO production were observed at higher electrical stimulation frequencies in association with declines in the positive amplitude of the contraction frequency response.^{116 117} The declines in amplitude of contraction at higher frequencies were reversed by administration of NOS inhibitors or methylene blue and could be mimicked with NO-donor drugs or 8-bromo-cGMP.¹¹⁶

In early studies of isolated adult rat cardiac myocytes, it was observed that inhibition of endogenous eNOS activity did not affect baseline contractile shortening but did potentiate the contractile response to submaximal concentrations of the ß-adrenergic agonist isoproterenol.¹⁷¹ This suggested that the agonist-regulated production of NO could act as a physiological counterregulator to limit the positive inotropic effect of adrenergic agonists. Similar results were obtained in dogs and in humans with heart failure (where eNOS has been identified in cardiac myocytes by immunohistochemistry; see above), in which myocardial NOS inhibition resulted in potentiation of the contractile responses to isoproterenol and dobutamine.^{172 173}

In studies of isolated ventricular myocytes from neonatal rats, the negative chronotropic effect of muscarinic cholinergic agonists was abrogated by NOS inhibition.¹⁷¹ By contrast, the effects of muscarinic cholinergic activation were not mediated by NO in isolated ventricular myocytes from the guinea pig¹⁷⁴ and atrial myocytes from the frog,¹⁷⁵ which do not appear to express any constitutive NOS activity. NOS inhibitors abolished the muscarnic cholinergic attenuation of isoproterenol-stimulated increases in L-type calcium current intensity and amplitude of contraction (the classic "accentuated antagonism") in specialized atrioventricular conduction cells and ventricular myocytes from adult rabbits and rats, respectively, all of which were shown to express eNOS.^{81 85} More important, experiments using intracoronary infusions of NOS inhibitors in open-chest dogs were also consistent with the interpretation that NO synthesis mediated the antagonistic effect of parasympathetic stimulation on the contractile responses elicited by dobutamine or isoproterenol.¹⁷⁶ Additional work indicated that sustained increases in cAMP induced within rat ventricular myocytes in vitro or achieved in vivo by administration of milrinone, the type 3 phosphodiesterase inhibitor, were accompanied by downregulation of eNOS mRNA, protein, and enzyme activity; these changes were accompanied by an increase in contractile responsiveness to ß-adrenergic agonists and a reduction in responsiveness to muscarinic cholinergic agonists.⁹⁵ The fact that downregulation of eNOS as mentioned above was only observed in cardiac myocytes and not in endothelial cells from the same hearts has the advantage of allowing the potentiation of adrenergic contractile responsiveness while maintaining the physiological regulation of coronary vascular tone through endothelium-derived NO. It will be of interest if future studies correlate cAMP-induced decreases in eNOS-dependent muscarinic signaling of isolated cardiac myocytes with the known alterations of the parasympathetic regulation of heart function in pathophysiological situations, such as heart failure, where the hyperadrenergic drive would be expected to increase intracellular cAMP.

As discussed previously, cytokines can also induce NO-independent¹⁷⁷ and NO-dependent¹⁷⁸ effects on myocardial contractility that are unrelated to iNOS gene expression. In studies of papillary muscles, one group of investigators observed negative inotropic effects of recombinant TNF-, IL-2, and IL-6, which occurred within minutes (before possible induction of iNOS) and were reversed by L-N-monomethyl-arginine (L-NMMA), an NOS inhibitor.¹⁷⁸ The mechanism of these apparently NO-mediated effects is not known but may involve activation by cytokines of a constitutively expressed NOS (probably eNOS) within cardiac muscle.¹⁷⁸ NO-independent effects of TNF- to activate the neutral sphingomyelinase pathway may also contribute to its negative inotropic effects.¹⁷⁹

	Biological Roles of NO Produced by iNOS: Paracrine and Autocrine Effects

Top Abstract Introduction NOS nNOS iNOS eNOS Molecular Targets of NO Biological Effects of... Biological Effects of Myocardial... Biological Roles of NO... Mechanisms by Which NO... References

 
In cardiac inflammation such as viral myocarditis, myocardial infarction, and cardiac allograft rejection, iNOS has been identified in endothelial cells, vascular smooth muscle cells, infiltrating macrophages and lymphocytes, and cardiac myocytes^{180 181 182} (Fig 2). When rat coronary microvascular endothelial cells were treated with IL-1ß, iNOS mRNA, protein, and enzyme activity were induced.^{55 67} When freshly isolated rat cardiac myocytes were added to cultured IL-1ß–treated microvascular endothelial cells and studied by videomicroscopy, the basal contractile function of the myocytes was not affected; however, the contractile response to isoproterenol was reduced, and this reduction was reversed by NOS inhibition.⁶⁷ Thus, activation of endothelial cell iNOS by cytokines can depress contractile responsiveness of adjacent cardiac myocytes. Depressed contractile responses of heart muscle have also been observed in pathological settings such as myocardial infarction and allograft rejection, when there is abundant expression of iNOS in macrophages infiltrating the myocardium and, to a lesser extent, in cardiac myocytes.^{180 182 183} In addition, exposure of isolated paced adult rat cardiac myocytes to media from LPS-conditioned activated macrophages produced attenuation of the inotropic response to isoproterenol, which was abrogated by an NOS inhibitor.⁴⁶ Subsequent studies indicated that this effect of the conditioned media was due to induction of iNOS in the cardiac myocytes themselves.⁵⁴

Impaired cardiac contraction is a known complication of the hypotensive syndrome associated with Gram-negative sepsis and can be observed in the absence of significant myocyte necrosis (for a review, see Reference 184¹⁸⁴ ). LPS was shown to induce iNOS activity and impair contraction in guinea pig hearts, an effect that was confirmed in isolated myocytes from the same animals.¹⁸⁵ Several other groups of investigators found that treatment with LPS or cytokines such as TNF-, IFN-, and IL-1ß induced iNOS mRNA, protein, and NO synthesis in rat cardiac myocytes^{44 45 54 180} and that induction of NOS 2 was associated with diminished inotropic responsiveness to ß-adrenergic agonists.^{46 54} In isolated, perfused, working rat hearts, administration of IL-1ß and TNF- was associated with a decline in contractile function that was partially ameliorated by treatment with an NOS inhibitor.¹⁸⁶ A similar improvement of left ventricular contractile function¹⁸⁷ or overall survival¹⁸⁸ has been observed after NOS inhibition in endotoxemia.

The induction by cytokines of high-output NO synthesis by iNOS in macrophages and other cells and the constitutive expression of iNOS in bronchial and intestinal mucosa are observations that are consistent with a role for this enzyme in natural immunity to infection by viral, mycobacterial, and parasitic pathogens.^{2 3} Evidence for such a protective role for iNOS in the heart has been provided by studies of NOS 2–knockout mice that succumbed more rapidly to infection or endotoxin.^{189 190} However, in some pathologic settings such as myocardial infarction or cardiac allograft rejection, NO production by iNOS may be deleterious and associated with contractile dysfunction and death of cardiac myocytes.^{140 180 182 191 192} Electron paramagnetic spin resonance (EPR) spectra that were consistent with NO interaction with iron-sulfur clusters in heart cells were observed in rejecting rat cardiac allografts.¹⁹³ Treatment of rat cardiac allografts with the partially selective inhibitor of iNOS, aminoguanidine, was associated with improved contractile performance of papillary muscles, prolonged allograft survival, and milder histopathology.¹⁸³ Induction of iNOS by LPS and IFN- in macrophages cocultured with adult rat cardiac myocytes was associated with increased myocyte death (assessed by creatine kinase release and trypan blue exclusion) that was partially reversed by NOS inhibition.¹⁹⁴ Similarly, induction of iNOS by TNF-, IL-1ß, and IFN- in isolated, purified, adult rat cardiac myocytes was associated with an NO-dependent increase in cell death.¹⁹⁴

Other studies have indicated that NO donors can trigger apoptosis in macrophages, vascular smooth muscle cells, and in preliminary experiments, in cardiac myocytes.^{195 196 197 198 199} Studies in vivo have shown that apoptosis of macrophages and cardiac myocytes occurs in parallel with iNOS induction in experimental models of cardiac allograft rejection and myocardial infarction and in endomyocardial biopsies from rejecting human hearts.^{140 182 200} The available data are consistent with the concept that in some settings, NO produced by iNOS may diminish cardiac function by increasing cell death, possibly by triggering apoptosis. Immunohistochemical evidence for nitrated tyrosine residues on cardiac myocyte cell membranes in these settings suggests that peroxynitrite formed from the interaction of NO and superoxide, along with hydroxyl breakdown products, may also contribute to cell damage in these settings.^{138 140} Of interest, high circulating levels of cytokines such as TNF- ²⁰¹ have been observed in patients with heart failure, and iNOS activity^{202 203} as well as iNOS and TNF- proteins localized by immunohistochemistry^{190 191 204} have been demonstrated in sections of myocardium from patients with dilated cardiomyopathy. These associations and the observation of apoptotic myocytes in the human failing myocardium²⁰⁵ all concur to suggest but do not prove a role for iNOS in the pathogenesis of these clinical syndromes.

	Mechanisms by Which NO Regulates Cardiac Function

Top Abstract Introduction NOS nNOS iNOS eNOS Molecular Targets of NO Biological Effects of... Biological Effects of Myocardial... Biological Roles of NO... Mechanisms by Which NO... References

 
The molecular mechanisms by which NO influences myocardial performance are the subject of much investigation but remain largely unexplored (Fig 3). Given the well known action of NO to stimulate soluble guanylyl cyclase, much attention has been paid to cGMP-activated pathways.¹¹⁹ NO and cGMP can decrease cardiac myocyte L-type calcium current and contraction through activation of cGMP-stimulated cAMP phosphodiesterase (type 2 PDE). This would decrease intracellular cAMP levels and the activity of PKA which in turn would alter the phosphorylation state of several target proteins, including the -subunit of the L-type calcium channel. Evidence for NO's effect mediated through activation of the cGMP-stimulated cAMP type 2 PDE to decrease myocyte contraction and/or L-type calcium current was provided in sinoatrial and atrioventricular myocytes from the rabbit,^{85 206 207} ventricular myocytes from the rat²⁰⁸ (all via muscarinic cholinergic activation of endogenous eNOS), and atrial myocytes from the frog²⁰⁹ (the latter via perfusion with micromolar concentrations of the NO donor 3-morpholino syndnonamine [SIN-1]). In all these experiments, the effect of NO was mainly to antagonize the actions of previously elevated cAMP; there was little, if any, effect of NO on the basal contractile state. The involvement of PDE 2 was inferred from the lack of efficacy of NO to antagonize the effects of the phosphodiesterase inhibitor isobutyl methylxanthine or nonhydrolyzable 8-bromo-cAMP.

View larger version (21K): [in this window] [in a new window]   Figure 3. Second-messenger pathways for the action of NO in cardiac myocytes. ß-Adrenergic receptor stimulation produces a positive inotropic effect through G protein -s–coupled activation of adenylyl cyclase, leading to increases in intracellular cAMP levels. Subsequent activation of cAMP-dependent protein kinase leads to downstream phosphorylation of target proteins, including those on the L-type calcium channel. The resultant increased influx of Ca2+ activates intracellular calcium-induced calcium release and enhances myofibrillar contraction. iNOS gene transcription and protein expression is induced in cardiac myocytes on exposure to cytokines or other inflammatory mediators. eNOS activation in cardiac myocytes occurs in response to both muscarinic cholinergic or adrenergic receptor stimulation through unidentified mechanisms. NO produced by either eNOS or iNOS activates the soluble isoform of guanylyl cyclase to increase intracellular levels of cGMP, which opposes the positive inotropic effects of cAMP through (1) activation of the cGMP-stimulated phosphodiesterase (PDE type 2) to enhance the breakdown of cAMP (conversely, cGMP may potentiate the effects of cAMP through inhibition of PDE type 3); (2) activation of the cGMP-dependent protein kinase (PKG), leading to downregulation of the L-type calcium current via either direct phosphorylation of the channel or phosphorylation of an intermediate protein opposing the effect of PKA. PKG also decreases myofilament sensitivity to calcium, thereby promoting relaxation (not shown). In addition, NO may affect myocyte contraction through mechanisms independent of cGMP elevations (see the Table). Aside from stimulating NO production, muscarinic cholinergic receptor stimulation results in Gi ß,–mediated inhibition of adenylyl cyclase and activation of distinct K+ channels, all of which variably participate in the parasympathetic inhibition of cardiac myocyte function depending on the species and region of the heart. AC indicates adenylyl cyclase; M-chol, muscarinic cholinergic receptor; ß-Adr, ß-adrenergic receptor; GC, guanylyl cyclase; PDE, phosphodiesterase; PKG, cGMP-dependent protein kinase; and PKA, cAMP-dependent protein kinase. See Fig 1 legend for explanation of other abbreviations.

Conversely, in some species, NO may potentiate the stimulatory effect of adrenergic stimulation or cAMP on cardiac myocyte contraction and L-type calcium current through inhibition of another cGMP-inhibited cAMP phosphodiesterase (type 3 PDE). This has been observed in frog atrial²⁰⁹ and adult rat ventricular²¹⁰ myocytes with low concentrations of the NO donors SIN-1 (<100 nmol/L) and S-nitroso-acetyl-penicillamine (SNAP) (<100 µmol/L), respectively. Similar concentrations of SIN-1 had no effect on basal calcium current in frog cells²⁰⁹ but increased it in human atrial myocytes, where basal cAMP levels might be higher.²¹¹

Higher concentrations of NO donors or cGMP analogues also activate PKG, which decreases calcium current intensity and contraction, especially after their initial stimulation after increases in cAMP.^{208 210 212 213 214} The same pathway is operative after muscarinic cholinergic stimulation in guinea pig²¹⁵ and rat ventricular²⁰⁸ myocytes. PKG activation may also decrease cardiac myocyte contraction through a desensitization of cardiac myofilaments for calcium¹⁸⁷ that is possibly related to phosphorylation of troponin I.

Some or all of the cGMP-dependent pathways mentioned above probably coexist in some cells,^{208 210 216} and the predominance of any one to produce the observed effect may vary according to the species or the region of the heart, as well as the stimulus and the experimental conditions used.

NO or its redox-related derivatives may also regulate channel function and cardiac contraction through mechanisms independent from cGMP (the Table). Peroxynitrite has been demonstrated to inhibit enzymes involved in the citric acid cycle, such as the interaction between cis-aconitase and iron-sulfur clusters.²¹⁷ In many systems NO and its derivatives, such as the nitrosonium ion (NO⁺), interact with sulfhydryl groups to produce biologically active S-nitrosoproteins,¹²⁰ which can then support additional transnitrosation reactions with other target sulfhydryl-containing proteins, the activity of which can be enhanced or impaired. By S-nitrosylating the glycolytic enzyme GAPDH, NO increases its auto-ADP-ribosylation and decreases its activity, thereby impeding glycolysis.^{132 218} NO has also been shown to reduce oxygen consumption in muscle slices, presumably by inhibiting mitochondrial electron transfer,^{219 220} an effect that was reproduced in neonatal rat ventricular myocytes treated with IL-1ß.²²¹ This effect could involve inactivation of the heme moiety of cytochrome c oxidase by NO.²²² Finally, studies of isolated, perfused hearts by nuclear magnetic resonance spectroscopy have suggested that NO released from S-nitrosoacetylcysteine, an NO donor, impaired augmentation of contractile performance in response to increased calcium, possibly by nitrosylating creatine kinase and impeding phosphoryl transfer from creatine phosphate to ATP.²²³ Further elucidation of the molecular signaling mechanisms that modify expression of the NOS isoforms and that determine the actions of NO will undoubtedly enhance understanding of the roles of endothelial cell and myocardial cell NO in the modification of cardiac function and in diseases of the heart.

View this table: [in this window] [in a new window]   Table 1. Molecular Targets of NO Relevant to Cardiovascular Biology

	Footnotes

 

Received February 20, 1997; accepted May 28, 1997.

	References

Top Abstract Introduction NOS nNOS iNOS eNOS Molecular Targets of NO Biological Effects of... Biological Effects of Myocardial... Biological Roles of NO... Mechanisms by Which NO... References

 

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