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(Circulation Research. 1996;79:363-380.)
© 1996 American Heart Association, Inc.

Articles

Nitric Oxide and Cardiac Function

Ralph A. Kelly, Jean-Luc Balligand, Thomas W. Smith

the Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Mass.

Correspondence to Ralph A. Kelly, MD, Cardiovascular Division, Brigham and Women's Hospital, 75 Francis St, Boston, MA 02115. E-mail rakelly@bics.bwh.harvard.edu.

Key Words: endothelial nitric oxide synthase • inducible nitric oxide synthase • signal transduction • autonomic nervous system • heart failure

	Introduction

Top Introduction NOSs NOSs in the Heart Role of NOx in... References

 
Over the past 5 years there has been an explosion of new information on the physiological and pathophysiological roles of NO within the heart as in other organ systems. In this review, we focus primarily on the regulation of cardiac contractile function by NO. We do not cite many relevant earlier articles (eg, articles on the regulation of cardiac contractility by cyclic nucleotides) that can be accessed directly from references in the manuscripts that are cited, and we ask the forbearance of colleagues whose work has not been cited because of space constraints.

We begin with a brief description of the biochemistry of NO and of each of the three NOS isoforms that have been characterized to date. This is followed by a more detailed overview of the physiological roles of iNOS (or NOS2) and eNOS (or NOS3) in cardiac muscle. We end with a brief review of the effects of pharmacological donors of NO on I_Ca-L and of possible physiological actions of NO and related congeners that are not thought to be mediated via activation of guanylyl cyclase.

	NOSs

Top Introduction NOSs NOSs in the Heart Role of NOx in... References

 
The free radical gas, NO, is generated by a family of enzymes known as NOSs that catalyze the conversion of the cationic amino acid L-arginine to L-citrulline in the presence of O₂ and NADPH. Depending on the redox status of the cell and the availability of transition metals such as heme iron, low-molecular-weight and -protein thiols, and oxygen-derived free radicals, a number of bioactive derivatives of NO can be generated, each with specific chemical and pharmacological properties that we will collectively term NO_xs.^{1 2 3 4 5} To date, three NOSs have been described, each the product of separate genes that share 50% to 60% amino acid homology.⁶ All three isoforms, each of which is presumed to function as a homodimer during activation, share a carboxy-terminal reductase domain homologous to the cytochrome P-450 reductases and an amino-terminal oxygenase domain containing a heme prosthetic group, which are linked roughly in the middle of the protein by a calmodulin-binding domain. Binding of calmodulin appears to act as a "molecular switch" to enable electron flow from flavin prosthetic groups in the reductase domain to heme, thereby facilitating the conversion of O₂ and L-arginine to NO and L-citrulline.^{7 8 9 10} The reductase domain of each NOS isoform also contains an H₄B prosthetic group, and H₄B is required for efficient generation of NO.¹¹ Unlike other enzymes, such as aromatic amino acid hydroxylases, where H₄B is used as a source of reducing equivalents and is recycled by dihydrobiopterin reductase, in the NOSs H₄B appears to be necessary to maintain a stable conformation for electron transport, possibly by promoting homodimerization.^{12 13 14 15} In addition, the heme prosthetic group appears to be necessary for H₄B binding and enzyme dimerization.¹⁶

Although originally classified as "constitutively expressed" and "Ca²⁺ sensitive," the NOS isoforms first described in neuronal tissue (nNOS or NOS1) and large vessel endothelial cells (eNOS or NOS3) are now known to be present in a number of cell types and to exhibit regulated expression under specific physiological conditions.^{17 18} In NOS1 and NOS3, physiological concentrations of Ca²⁺ in cells regulate the binding of calmodulin to the "latch domain," thereby initiating electron transfer from the flavins to the heme moieties. In contrast, calmodulin remains tightly bound to the "inducible" and "Ca²⁺-insensitive" isoform termed iNOS or NOS2 even at low physiological intracellular Ca²⁺ levels, acting essentially as a subunit of this isoform.^{7 8 9 10} Although this is true at physiologically relevant intracellular Ca²⁺ activity, chelation of Ca²⁺ does result in dissociation of calmodulin and loss of enzyme activity. The Ca²⁺- and calmodulin-binding domains have been identified for NOS2 and NOS3 (ie, iNOS and eNOS), and functional chimeric proteins have been created, eg, a Ca²⁺/calmodulin-regulated NOS2 chimera.¹⁹ Importantly, the availability of L-arginine may be rate limiting for NOS activity in some cell types, particularly after induction of NOS2, a point that will be discussed in more detail below with reference to cardiac myocytes.²⁰

NO itself may regulate NOS expression and activity.²¹ Both NOS1 and NOS2 have been shown to form ferrous-nitrosyl complexes in their heme prosthetic groups that may act to partially self-inactivate these enzymes under certain conditions.^{22 23} Increased production of NO_x by cytokine-activated macrophages has been demonstrated to reduce heme availability and insertion into newly synthesized NOS2 monomers, preventing dimer formation and enzyme activity.²⁴ Increased intracellular cGMP levels in pulmonary vascular endothelial cells after activation of NOS3 by NO_x have also been shown to increase NOS expression in these cells, whereas authentic NO in solution and NO donors have been documented to reduce NOS2 expression in activated endothelial cells, possibly by interfering with NF-B signaling.^{25 26 27}

All three NOS isoforms appear to be spatially constrained within some cell types that express these enzymes. NOS1, originally characterized as cytosolic in neuronal tissue, is now known to be complexed with PSD-95 and PSD-93 in neuronal tissue and with the cytoskeletal protein 1-syntrophin, a protein in the dystrophin complex in fast-twitch skeletal muscle, through an amino-terminal cytoskeletal protein-binding domain that is unique to this isoform.^{28 29 30} Although originally presumed to be localized to the cytosol, NOS2 also has been detected in a particulate fraction in primary isolates of activated murine peritoneal macrophages. Neither the posttranslational modification(s) that leads to association with membranes nor the specific membrane compartment has yet been characterized.³¹

Unlike NOS1 or NOS2, NOS3 undergoes dual acylation by myristic and palmitic acid, posttranslational modifications that result in targeting of this isoform to cell membranes. N-myristoylation appears to be irreversible and necessary for membrane association, whereas palmitoylation of specific amino-terminal cysteines appears to be reversible and may be dynamically regulated under physiological conditions.^{32 33 34} After binding of agonists such as bradykinin in endothelial cells, NOS3 undergoes depalmitoylation with translocation to the cytosol as well as phosphorylation, both of which are associated with enzyme activation.^{33 34} Recombinant NOS3 also undergoes phosphorylation by PKA, PKC, and Ca²⁺-calmodulin kinase.³⁵ NOS3 within endothelial cells is also known to be phosphorylated in response to agonists such as bradykinin, and this correlates with translocation from a membrane-associated (particulate) fraction to a cytosolic (soluble) fraction. The importance of these posttranslational modifications by protein kinases is still unclear.

The specific "membrane" compartment(s) associated with acylated NOS3 has been controversial.³⁶ Since dual acylation is a common posttranslational modification of some cellular signaling molecules (eg, GTP-binding [G] protein subunits) associated with NOS3 activation, it is reasonable to assume that these spatial restraints play a role in cellular signal transduction pathways. This is of relevance to cardiac myocytes, which also express NOS3, most of which (as in endothelial cells) can be found in a particulate membrane-associated fraction (see below).

In endothelial cells, acylated NOS3 is now known to be localized to caveolae, plasmalemmal microdomains that are known to facilitate the transcytosis of macromolecules and the uptake of small molecules by potocytosis.^{37 38} Caveolae, and associated DIGs, have also been implicated in the compartmentalization of proteins involved in specific signal transduction cascades, including, among others, tissue factor, platelet-derived growth factor receptors, PKCs, muscarinic cholinergic receptors, multiple heterotrimeric G proteins, an inositol trisphosphate–regulated Ca²⁺ channel, and the plasmalemmal Ca²⁺-ATPase.^{39 40 41 42} The 21- to 24-kD protein vesicular integral membrane protein 21, or caveolin-1, the principle scaffolding protein that forms the caveolar "coat," has been shown to directly inhibit GDP/GTP exchange of G protein subunits and can be phosphorylated directly by Src family tyrosine kinases, implying that caveolins themselves may participate in the regulation of some signal transduction mechanisms.^{44 45} Caveolin-1 has also been shown to interact selectively with wild-type H-Ras but not with a mutationally activated soluble H-Ras, suggesting that caveolins may facilitate the binding of specific G proteins depending on their activation state.⁴⁶ Striated muscle, including cardiac muscle in which caveolae are abundant, has been shown to express a unique caveolin isoform, known as caveolin-3, which exhibits both GTPase activating protein–like activity and, at higher concentrations, GTPase inhibitory activity.⁴⁷ Both myristoylation and palmitoylation are necessary to efficiently target NOS3 to DIGs and caveolae in endothelial cells and transfected COS cells.³⁷ Knowledge of the cell physiology of each of the NOS isoenzymes in many cell types will be further enhanced as the role of caveolae and DIGs in intracellular protein trafficking and, along with other scaffolding and anchoring proteins, in the compartmentation and regulation of specific signal transduction cascades becomes better understood.⁴⁸

	NOSs in the Heart

Top Introduction NOSs NOSs in the Heart Role of NOx in... References

 
NOS1: "Brain" or "Neuronal" NOS
The anatomic distribution and physiological role(s) of NOS1 in the heart have been the subjects of relatively few reports to date. Unlike some skeletal muscle fibers, cardiac myocytes do not appear to express NOS1.^{28 29 49 50} Early reports did demonstrate the existence of NADPH-diaphorase–positive histochemical staining (a relatively insensitive and somewhat nonspecific marker for NOS) in intrinsic neurons within the heart.^{51 52 53} These studies have since been verified by selective staining with NOS1 antibody preparations.^{49 54 55} Most NOS1 staining has been identified in the atria, along epicardial coronary arteries, and in specialized cardiac conduction tissue such as the sinoatrial and atrioventricular nodes.^{49 51 52 53 54 55 56 57 58} Colocalization of NOS1 with tyrosine hydroxylase has been noted in some neurons, indicating that some sympathetic postganglionic neurons in the heart appear to express this isoform.^{49 58} However, NOS1 staining was also present in many neurons that were negative for tyrosine hydroxylase, suggesting that cholinergic as well as a subset of nonadrenergic noncholinergic neurons (termed "nitrergic" by Rand and Li⁵⁹ ) may also express other NOS isoforms. Finally, NOS3 has also been identified in neurons in the brain and in stellate ganglia, indicating that this NOS may also participate in neuronal signaling.^{17 57}

The physiological roles of neuronal NO in the heart are less clear. Schwarz et al⁴⁹ have reported that both NO derived from pharmacological NO donors and endogenous NO_x largely derived from NOS1 in intracardiac sympathetic neurons decrease norepinephrine release in Langendorff-perfused adult rat hearts during electrical sympathetic nerve stimulation. The stimulation frequencies used were sufficiently low to exclude an important role for autoinhibition of norepinephrine release by activation of preganglionic ₂-receptors. These investigators also excluded endothelial cell–derived NO_x in this model, since perfusion of the heart with a nondenaturing detergent to damage selectively the coronary endothelium had no effect on norepinephrine release. Also, desipramine had no effect on rates of norepinephrine release in this model. However, Kaye et al,⁵⁸ in a preliminary report, noted that both pharmacological donors of NO and autocrine or paracrine endogenously generated NO_x decreased desipramine-inhibitable uptake of norepinephrine (ie, uptake-1) into sympathetic neurons or a rat pheochromocytoma cell line (PC12). Since there have been several reports of the inhibition of biogenic amine release and reuptake by NO in the central nervous system, it is possible that catecholamine signaling is regulated by both mechanisms in sympathetic nerves in the heart.^{49 57 60} Finally, Horackova et al⁶¹ reported that either an NO donor or L-arginine increased the spontaneous beating rate of adult guinea pig ventricular myocytes in long-term coculture with primary stellate ganglion or "intrinsic cardiac" neurons. Among other mechanisms, these data are consistent with the hypothesis that exogenous or endogenous generation of NO_x could diminish uptake-1–mediated catecholamine transport.

NOS2: "Cytokine-Inducible Ca²⁺-Insensitive" NOS
In 1992, shortly after the first publications by Xie et al⁶² and Lowenstein et al⁶³ of the cloning and characterization of an NOS expressed in activated murine macrophages, reports by Schulz et al,⁶⁴ Roberts and colleagues,^{65 66} Balligand et al,⁶⁷ and Brady et al⁶⁸ provided the first functional and biochemical evidence for a cytokine- and LPS-inducible Ca²⁺-independent NOS in cardiac myocytes. Schulz et al⁶⁴ demonstrated that both ventricular muscle isolated from rats pretreated with LPS in vivo and isolated adult rat ventricular myocytes pretreated in vitro with the cytokines IL-1ß and TNF exhibited a time-dependent and dexamethasone-inhibitable increase in Ca²⁺-independent enzymatic activity and, in the production of nitrite and nitrate, stable oxidative metabolites of NO_x (ie, NO_x^-). Roberts et al⁶⁵ observed that IL-1ß alone decreased the spontaneous beating rate of neonatal rat ventricular myocytes in vitro concurrent with an L-arginine–dependent increase in the release of NO_x into IL-1ß–treated myocyte-conditioned medium. These actions of IL-1ß could be prevented by TGFß.^{65 66} Brady et al⁶⁸ noted that electrically stimulated adult guinea pig ventricular myocytes, obtained from animals pretreated 4 hours earlier with LPS, exhibited a depressed amplitude of shortening compared with myocytes from control animals. This effect of LPS on myocyte contractile function could be abolished by pretreatment of animals in vivo with dexamethasone or reversed by the addition of an L-arginine analogue NOS inhibitor in vitro. Reports from a number of laboratories have confirmed and extended these observations, and these will be described below in two contexts: (1) the regulation of NOS2 expression and activity and (2) the potential functional consequences of NOS2 induction in the heart. It should be noted that both LPS and the cytokines discussed in the present review, alone and in combination, are known to have many actions in addition to the induction of NOS2 that will affect the phenotype and function of cardiac muscle cells.

In addition to LPS, IL-1ß, and TNF, IFN and IL-6 have been shown to induce NOS2 mRNA and activity in cardiac myocytes.^{67 68 69 70 71 72 73 74 75 76} Among the other cellular constituents of cardiac muscle, NOS2 has been detected in vivo and in vitro in endocardial endothelium, infiltrating inflammatory cells (presumably macrophages), vascular smooth muscle, fibroblasts, and the microvascular endothelium, and in cardiac myocytes.^{64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82} For the present review, we will focus on the regulation of NOS2 expression in cardiac myocytes and microvascular endothelial cells, which together, with the possible exception of infiltrating inflammatory cells, probably account for the majority of NO_x production following global or regional NOS2 induction in the myocardium.^{69 77} NOS2 mRNA can be detected by Northern blot within 4 to 6 hours, and NOS2 protein and activity can be detected within 6 to 12 hours in primary isolates of adult rat ventricular myocytes and confluent serum-starved primary cultures of cardiac microvascular endothelial cells after exposure to recombinant human IL-1ß and TNF and recombinant murine IFN.^{64 69 77} Induction of NOS2 is diminished in both cell types by either dexamethasone or TGFß.^{64 65 69 77} NOS2 is readily detectable by immunohistochemistry in microvascular endothelial cells in vivo in experimental animals after intraperitoneal injection of LPS, in heterotopically transplanted cardiac allografts, and in vitro after cytokine exposure.^{77 78 79 80} NOS2 staining in cardiac myocytes is either variable or undetectable using standard immunohistochemical techniques in similar animal models and in vitro. This likely reflects the lower amounts of NOS2 protein present within cardiac myocytes relative to adjacent endothelial cells, smooth muscle cells, and tissue macrophages. Confirmation that detectable NOS2 mRNA and protein, on Northern and Western blots, respectively, and the generation of NO_x in primary cultures of adult rat ventricular myocytes were indeed of myocyte origin was obtained by Balligand et al.⁶⁹ NO released from single, isolated, cytokine-pretreated myocytes could be detected using the porphyrnic/Nafion-coated microsensor originally described by Malinski and Taha.⁸³

The intracellular signal transduction pathways regulating NOS2 induction and activity in ventricular myocytes and microvascular endothelial cells that have been most intensively examined to date are those for IL-1ß and IFN. IL-1ß alone induces NOS2 in both cell types, whereas IFN alone only induces NOS2 in cardiac myocytes. Nevertheless, IFN enhances the rise in NOS2 mRNA and protein in both cell types in response to IL-1ß.^{69 72 77} As has been observed in other cell types, including vascular smooth muscle, induction of NOS2 by either cytokine is preceded by activation of p44/p42 MAPKs (ERK1/ERK2)⁸⁴ (Fig 1). Although IFN does not activate these MAPKs in cardiac microvascular endothelial cells in vitro, it does initiate STAT1 phosphorylation and translocation to the nucleus in these cells and in cardiac myocytes. Both the human and rodent NOS2 promoters contain AP-1 and GAS sequences, consistent with MAPK and STAT-mediated signaling.^{85 86} Both PKC desensitization with phorbol esters and the diacylglycerol-selective PKC isoenzyme inhibitor bisindolylmaleimide inhibit NOS2 induction by IL-1ß in cardiac myocytes but not in microvascular endothelial cells.⁸⁴ LPS has also been shown to induce NOS2 in ventricular myocytes by activating a PKC-dependent pathway.⁸⁷

View larger version (21K): [in this window] [in a new window]   Figure 1. NOS2 induction in cardiac myocytes. Illustrated are the signal transduction cascades presumed to be involved in the induction of NOS2 transcription, as described in References 84 and 223. For clarity, the NF-B–mediating signaling pathway has been omitted from this figure. ERK1/ERK2 indicates p44/p42 MAPKs; MEK1/MEK2, MAPK kinases; BIM, bisindolylmaleimide, an inhibitor of diacylglycerol-activated PKCs; BZA-5B, an inhibitor of farnesyl transferase; and JAK, Janus kinase.

An understanding of the complex cellular and subcellular mechanisms that regulate NOS2 expression has just begun to emerge. An analysis of the human NOS2 promoter has revealed multiple cytokine-responsive elements up to 16 kb 5' to the transcription start site.⁸⁸ In addition, neurotransmitter and peptide autacoid signaling pathways that involve activation of specific PKC isoforms have also been shown to enhance NOS2 transcription and/or activity. Ikeda and colleagues^{89 90} have shown that both angiotensin II and the -adrenergic agonist phenylephrine augment cytokine-induced NOS2 expression in cardiac myocytes. In addition, agents that increase intracellular cAMP or membrane-permeant cAMP analogues augment NOS2 mRNA levels in cardiac myocytes in response to IL-1ß, largely because of an enhancement in mRNA stability.^{91 92 93 229}

In addition to NOS2, cytokines also increase the expression of other proteins that enhance NOS2 activity in cardiac myocytes. Myocytes isolated from animals that had had an intraperitoneal injection of LPS exhibited coordinate upregulation of NOS2 and GTP cyclohydrolase I, the rate-limiting enzyme for de novo synthesis of H₄B.^{69 230 231} NOS2 induction by IL-1ß and IFN in neonatal and adult rat ventricular myocytes is also paralleled by increases in mRNAs and activities of the high-affinity CATs (CAT-1 and CAT-2B), as well as the low-affinity CAT (CAT-2A), responsible for L-arginine transport.²⁰ In addition, insulin, which has no effect on NOS2 mRNA or protein levels in cardiac myocytes, increases NO_x production in these cells by augmenting IL-1ß–induced and IFN-induced expression of CAT-1.^{20 72} Lysine, which in the presence of physiological concentrations of Na⁺ acts as an inhibitor of L-arginine transport by cationic amino acid transporters (ie, the "y⁺" phenotype transporter), diminishes cytokine- and insulin-mediated NO_x production by cardiac myocytes following NOS2 induction. These data imply, but by themselves do not prove, that intracellular L-arginine content is rate limiting for the production of NO_x by NOS2 in these and perhaps other cells. This interpretation is supported by the observation that the K_m for L-arginine in LPS-activated macrophages is 100 µmol/L, which is much higher than that reported for isolated enzyme preparations but close to both the K_m of the high-affinity CATs (CAT-1 and CAT-2B) for L-arginine and the plasma concentration of this amino acid (also 100 µmol/L).^{20 94}

Glucocorticoids, which are known to interrupt cytokine-dependent signaling at multiple sites in most cell types,²³¹ also increase expression of the multifunctional extracellular matrix phosphoprotein osteopontin by both cardiac myocytes and microvascular endothelial cells (Fig 2).⁹⁵ This was unexpected, since glucocorticoids decrease osteopontin expression in osteoblasts, the cell type in which the protein was first characterized. Osteopontin is produced in many tissues in response to inflammation or injury, including the vasculature and myocardium.⁹⁶ Osteopontin has been shown to decrease NOS2 expression in cytokine-pretreated primary cultures of renal epithelial cells and of cardiac myocytes and microvascular endothelial cells isolated from adult rat ventricular muscle.^{95 97} The mechanism by which increased synthesis of osteopontin decreases NOS2 expression is unknown but is mediated in part by interactions with the extracellular matrix, since interruption of integrin binding in vitro in cytokine-pretreated cardiac myocytes and microvascular endothelial cells increases NOS2 expression in both cell types.⁹⁵

View larger version (26K): [in this window] [in a new window]   Figure 2. Glucocorticoid-induced expression of osteopontin (OPN) suppresses NOS2 induction in cardiac myocytes and microvascular endothelial cells. Several mechanisms by which glucocorticoids (GCs), after association with their intracellular receptor proteins (RECs), are presumed to regulate expression of NOS2 are shown in this figure.224 225 226 227 This includes the induction of OPN expression, synthesis, and secretion, as described in Reference 95. After association with vß3 integrins on myocyte and microvascular endothelial cell membranes, NOS2 induction is suppressed by an as-yet-unknown sequence of events, possibly involving focal adhesion kinase (FAK).

NOS3: "Endothelial Constitutive" NOS
The NOS isoform originally characterized in large conduit vessel endothelium is now known to be expressed within the heart in the endothelium both of the endocardium and of the coronary vasculature (including capillary and venular endothelium), in cardiac myocytes, and in specialized cardiac conduction tissue (including sinoatrial and atrioventricular nodal tissue), as well as in some formed elements of the blood (including monocytes and platelets).^{50 98 99 100 101 102} The possibility that cardiac myocytes expressed a constitutively expressed NOS was suggested by Balligand et al¹⁰³ in a report from this laboratory in which L-arginine analogue NOS inhibitors were observed to regulate the spontaneous beating rate and contractile responsiveness of neonatal and adult rat ventricular myocytes, respectively, to muscarinic cholinergic and ß-adrenergic agonists. Indeed, Schulz et al⁶⁴ had demonstrated that adult rat ventricular myocyte primary isolates did contain Ca²⁺-sensitive NOS enzymatic activity, although contamination by nonmyocyte sources of NOS activity could not be excluded.

Subsequently, Balligand et al⁵⁰ reported an extensive characterization of the constitutive NOS activity in ventricular myocytes, documenting the absence of either NOS1 or NOS2 transcripts in highly purified myocyte primary isolates by RT-PCR using rat NOS isoform–specific amplimers and confirming the presence of NOS3 in these cells in vivo and in vitro by immunohistochemistry with NOS3-specific antibodies. Furthermore, the majority of NOS enzymatic activity was found in a particulate subcellular fraction after cell lysis and ultracentrifugation, suggesting that the myocyte constitutive NOS was largely associated with cellular membranes, a feature characteristic of NOS3 among the known NOS isoforms. Also, muscarinic cholinergic attenuation of ß-adrenergic agonist–stimulated L-type Ca²⁺ channel current and amplitude of shortening was blocked by intracellular dialysis of individual ventricular myocytes with either methylene blue or a specific L-arginine analogue NOS inhibitor, confirming the presence of a constitutive NOS activity linked to known signal transduction cascades in these cells. NOS3 mRNA has been detected in atrial and ventricular myocytes in sections of adult rat cardiac muscle and in human atrial myocytes by in situ hybridization.^{104 105} Finally, antibodies to caveolin-3, a striated muscle–specific caveolin as discussed above, are able to coimmunoprecipitate NOS3 from adult rat ventricular myocyte primary isolates (O. Feron, T.W. Smith, R.A. Kelly, and T. Michel, unpublished data, 1996). The expression of NOS3 in sinoatrial and atrioventricular nodal cells has recently been confirmed as well.¹⁰²

NOS3 was also identified in the microvascular endothelium of atrial and ventricular muscle, including capillaries and venules, as well as in the endothelium of resistance and epicardial coronary vessels and the endocardium.^{50 98 99} The physiological role and expression of NOS3 in capillary and venular endothelium would be expected to differ in part from those of resistance and muscular arteries and arterioles, since changes in enzymatic activity by shear stress, for example, would appear to be less physiologically relevant. Changes in NOS3 activity in the microvasculature could regulate platelet adhesion and vascular permeability, for example, and perhaps the function of subjacent cardiac myocytes. Interestingly, NOS3 expression is undetectable in cardiac microvascular endothelial cell primary cultures in standard conditions shortly after isolation, in contrast to conduit vessel endothelial cells, which continue to constitutively express this NOS isoform in culture, further highlighting differences in the regulation of NOS3 activity in these two endothelial cell phenotypes in culture. Sustained NOS3 expression can be maintained in both endothelial cell phenotypes, however, if seeded at high density in medium containing low concentrations of serum (Reference 106; L. Belhassen, J.-L. Balligand, T.W. Smith, and R.A. Kelly, unpublished data, 1996).

Little is yet known regarding transcriptional and posttranscriptional regulation of NOS3 in cardiac myocytes. The human NOS3 promoter is known to contain cis-regulatory consensus sequences for binding of transcription factors, including those for AP-1 and AP-2, retinoblastoma control element, shear stress response element, NF-1, steroid regulatory element-1, Sp-1, and a cAMP response element.^{107 108 109} Both the SR-1 and GATA elements have been reported to be essential for basal promoter activity in conduit vessel endothelium.^{108 109} Expression of NOS3 in both pulmonary arterial and aortic endothelial cells has also been shown to be regulated by oxygen. Liao et al¹¹⁰ have reported that an increase in oxygen concentration increased NOS3 mRNA abundance in primary cultures of these cells, whereas a decrease in oxygen concentration decreased NOS3 mRNA levels, apparently by both transcriptional and posttranscriptional regulatory mechanisms.

In a recent report from this laboratory, Belhassen et al¹¹¹ have shown that agents that increase intracellular cAMP in freshly isolated adult rat ventricular myocytes in vitro, such as 8-bromo-cAMP, forskolin, or IBMX, decrease NOS3 mRNA levels in these cells. This was probably due to a decline in NOS3 expression, since its mRNA half-life was unaffected. This effect could also be demonstrated in vivo, since ventricular myocytes isolated from adult rats that had been given milrinone in their drinking water for 3 days had no detectable NOS3 RNA or protein, by either Northern or Western blot analysis, respectively, or activity using standard biochemical assays for NOS. Furthermore, muscarinic cholinergic attenuation of ß-adrenergic agonist regulation of myocyte contractile function could no longer be demonstrated in myocytes from milrinone-pretreated animals (see below). Interestingly, NOS3 mRNA and proteins were still readily detectable in microvascular endothelial cells freshly isolated from ventricular muscle rats fed milrinone, indicating important differences in the regulation of NOS3 expression in these two cell types.¹¹¹

The posttranslational regulation of NOS3 activity in cardiac myocytes is the object of ongoing research in a number of laboratories. Although it is plausible that NOS3 in both cardiac myocytes and microvascular endothelial cells is myristoylated and palmitoylated like conduit endothelial NOS3, given its "particulate" distribution upon subcellular fractionation, possibly within caveolae, this has yet to be formally demonstrated.⁵⁰ As expected, myocyte NOS3 is activated by increases in intracellular Ca²⁺ activity, as has been demonstrated for this NOS in isolated adult rat ventricular myocytes during electric field pacing in vitro.¹¹² The rate of accumulation of nitrite (NO_x^-) in medium conditioned by these cells increases as a function of their pacing frequency and can be lowered to baseline levels by either a Ca²⁺ chelator (eg, BAPTA) or an L-arginine analogue NOS inhibitor.¹¹² Hattler et al¹¹³ have also demonstrated that net generation of NO_x across the coronary vascular bed declines markedly in human hearts in situ after cardiac arrest in patients undergoing cardiopulmonary bypass, implicating a link between contractile activity and NOx generation.

	Role of NOx in the Heart

Top Introduction NOSs NOSs in the Heart Role of NOx in... References

 
Physiological Effects of NOS2 Induction
As discussed above, a number of cellular constituents of cardiac muscle are now known to be capable of expressing NOS2 in response to LPS and specific cytokines, including the endothelium and smooth muscle of the cardiac microvasculature, the endocardial endothelium, tissue macrophages, and cardiac myocytes. The physiological roles and pathophysiological consequences following induction of this high-output NOS in these cells are less clear, however. The most convincing evidence for an important pathophysiological role for NOS2 to date has come from experimental animal models of the systemic inflammatory response syndrome and cardiac allograft transplantation, and these will be discussed in more detail below. Although a number of reports have appeared linking NOS2 induction to the pathogenesis of some forms of cardiomyopathy in humans, to date these have involved relatively small numbers of patients.^{114 115 116} Indeed, Thoenes et al¹¹⁷ could find no evidence of increased NOS2 expression in patients with heart failure in specimens obtained at postmortem, although NOS2 was present in the hearts of most of the patients who succumbed to systemic sepsis.

Nevertheless, recent reports in humans with cardiac allografts indicate that NOS2 expression is increased in the hearts of these patients and that serum levels of nitrite and nitrate are increased as well, consistent with experimental animal data reviewed below.^{118 119} Lewis et al¹¹⁸ also reported that those patients with evidence of NOS2 expression in endomyocardial biopsy specimens were more likely to have evidence of systolic and/or diastolic ventricular dysfunction as determined by echocardiography. Also, Haywood et al¹²⁰ have presented evidence that patients with heart failure were much more likely to have evidence of NOS2 in endomyocardial biopsy and surgical specimens than control (ie, necropsy or cardiac donor) specimens, as determined by several complementary techniques, including RT-PCR, Western blot, and immunohistochemistry. This increase in NOS2 expression was present in patients with heart failure whether it was due to idiopathic dilated cardiomyopathy, ischemic heart disease, or valvular heart disease. This report provides no direct evidence for a role for NOS2 in the pathogenesis of the heart failure syndrome. Nevertheless, if these data are confirmed, a role for induction and activation of this NOS isoenzyme in the pathophysiology of heart failure will need to be examined further.²²⁸ The evidence reviewed above indicating that NOS2 activity in cardiac myocytes is enhanced by increased intracellular cAMP or activation of PKC isoforms suggests that the elevated intracardiac or systemic levels of catecholamines and peptide autacoids, such as angiotensin II, that are characteristic of heart failure could promote and sustain NOS2 expression in this syndrome.

A role for NOS2-derived NO_x in myocardial contractile dysfunction has been studied in some detail in isolated cardiac myocyte preparations. Brady et al⁶⁸ initially reported that the depressed contractile function of guinea pig ventricular myocytes isolated from animals injected several hours earlier with LPS could be reversed by NOS inhibitors. Subsequently, in a report from this laboratory, Balligand et al⁶⁷ demonstrated that isolated paced adult rat ventricular myocytes that had been exposed to medium conditioned by LPS-activated rat alveolar macrophages exhibited a markedly reduced positive inotropic response to isoproterenol, a decline that could be rapidly reversed by NOS inhibitors. This depression in myocyte contractile responsiveness to ß-adrenergic agonists correlated with an increase in myocyte generation of NO_x and could be partly ameliorated by the addition of recombinant IL-1 receptor antagonist to the LPS-activated macrophage-conditioned medium. It could also be mimicked by the addition of species-appropriate recombinant cytokines including IL-1ß, TNF, and IFN.^{69 70} Pyo and Wahler¹²¹ have reported that ventricular myocytes isolated from rejecting cardiac allografts, but not isografts, exhibited a characteristic decline in contractile responsiveness to ß-adrenergic agonists in these cells, although the role of NO_x was not specifically addressed in this report. The addition of freshly isolated adult ventricular myocytes from normal rats to established confluent monolayer cultures of cardiac microvascular endothelial cells that had been pretreated with IL-1ß also exhibited decreased contractile responsiveness to ß-adrenergic agonists, indicating that generation of NO_x by NOS2 in the microvascular endothelium was sufficient to affect the function of cocultured myocytes.¹²²

Schulz et al¹²³ have also demonstrated that the combination of recombinant IL-1ß and TNF decreases the contractile function of isolated perfused working hearts with a time course consistent with induction of NOS2 by these cytokines. An L-arginine analogue NOS inhibitor, at concentrations that did not cause significant reductions in coronary flow, ameliorated the decline in contractile function. Other investigators have described similar reductions in cardiac myocyte contractile function several hours after exposure to LPS or recombinant cytokines, including IL-1ß and IL-6.^{73 124} Both the increase in NO_x production and the decrease in contractile responsiveness in LPS-treated adult rat cardiac myocytes was maximal at 6 hours and subsequently declined, unlike myocytes exposed to IL-1ß and IFN, which continued to produce NO_x.¹²⁴ In a chronically instrumented rat preparation, continuous intravenous infusion of LPS resulted in a peak in tissue NOS activity and blood oxidative NO_x metabolite content at 6 hours, followed by a decline, whereas the hemodynamic characteristics of systemic endotoxemia were sustained.¹²⁵ In contrast, Decking et al¹²⁶ reported that NOS2 inhibitors had no effect on the depressed contractile function of isolated perfused guinea pig hearts 4 hours after an intraperitoneal injection of LPS, although these authors could not detect any evidence of NOS2 enzymatic activity in these hearts at this time point. These results not only point to qualitative differences between the hemodynamic responses to chronic infusions of LPS and recombinant cytokines but also indicate that cellular mechanisms in addition to NOS2 induction are important in the pathogenesis of the systemic inflammatory response syndrome.¹²⁷ Interestingly, Ungureanu-Longrois et al⁷² from this laboratory noted that insulin was required for the decline in cytokine-pretreated myocyte contractile dysfunction to be manifested but not for the induction of NOS2 mRNA or protein. This may be explained in part by the observation that insulin enhances L-arginine transport into myocytes, as discussed above.²⁰

The physiological sequelae of NOS2 induction are not limited to a reversible decline in myocyte contractile function. Pinsky et al¹²⁸ have shown that adult rat ventricular myocytes exposed to either the combination of recombinant IL-1ß, TNF, and IFN or to an LPS- and IFN-preactivated murine macrophage cell line in coculture resulted in significant myocyte injury or death, as assessed by either creatine kinase release or a failure to exclude trypan blue. Addition of an L-arginine analogue NOS inhibitor or prevention of myocyte NOS2 induction by TGFß prevented the deleterious effects of the recombinant cytokines or of activated macrophages on myocyte survival. In a subsequent preliminary report, these investigators have provided evidence that some NO_x-dependent cytotoxicity may be due in part to induction of an apoptotic pathway in cardiac myocytes.¹²⁹

A sustained induction of NOS2 expression and generation of NO_x have been described by a number of laboratories in the rat heterotopic cardiac allograft model. Lancaster et al¹³⁰ reported that iron-nitrosyl electron paramagnetic resonance signals consistent with marked increases in NO_x generation could be detected in both blood and in rejecting allografts of rats with allogeneic heterotopic heart transplants but not in other organs or in cardiac allografts in animals in which acute rejection had been suppressed by administration of the immunosuppressant FK506. Yang et al,⁸⁰ Worrall et al,⁷⁹ Winlaw and colleagues,^{131 132} Kuo et al,¹³³ and Russell et al⁷⁸ have subsequently published evidence confirming the induction of NOS2 in inflammatory cells, the microvasculature, and cardiac myocytes in allogeneic but not syngeneic cardiac transplants. Russell et al demonstrated that allograft NOS2 mRNA levels peaked and then declined in the initial 2 weeks after transplantation. At the longer time points (ie, 2 to 4 months) at which concentric arteriosclerosis of the coronary arterial vasculature had become manifest, NOS2 staining by immunohistochemistry was now visible in smooth muscle cells in the media and to a lesser extent in the neointimal layers.

Worrall et al⁷⁹ also demonstrated that continuous intravenous infusions of the (somewhat) NOS2-selective inhibitor aminoguanidine improved graft survival and increased tension development in right ventricular papillary muscles obtained from allografted but not isografted hearts. Worrall et al¹³⁴ reported subsequently that microvascular permeability, as measured by the rate of permeation of radiolabeled albumin, was increased in rats with cardiac allografts, both in the graft itself and also systemically compared with isografted animals. This systemic and allograft vascular barrier dysfunction also could be ameliorated by systemic infusions of aminoguanidine, suggesting that generation of NO_x in allografted tissues can have systemic effects. These authors have also reported that dexamethasone effectively suppresses NOS2 induction in the allografted heart in this model.¹³⁵ Finally, Russell et al¹³⁶ have reported that a new therapeutic agent, the recombinant fusion protein CTLA4Ig, which limits T-cell activation by alloantigens among other actions, was more effective than cyclosporin A in decreasing NOS2 expression in long-term rat cardiac allografts.

Not all actions of NOS2 are necessarily deleterious. Nathan¹³⁷ has included the generation of NO_x by NOS2 in the category of "innate" immune responses, a phylogenetically primitive but rapidly activated form of host defense that can be brought to bear on invading organisms long before the highly selective "adaptive" immune response that involves clonal amplification of specific major histocompatibility complex (or MHC)–restricted lymphocytes. With the exception of IFN, a T-cell–derived cytokine that accelerates NOS2 induction in many cell types, the cytokines most closely associated with NOS2 induction (TNF, IL-1ß, and IL-6) are those typically characterized as mediators of innate or "natural" immunity. NO_x generated by NOS2 has been shown to suppress viral replication or reactivation and to promote bacterial killing.^{138 139 140} In both initial reports on the phenotype of mice lacking functional NOS2 genes (ie, NOS2 "knockouts"), the animals did not develop a fatal systemic inflammatory response–like syndrome after intraperitoneal injections of LPS but were susceptible to lethal infection with facultative intracellular and opportunistic pathogens at rates much higher than the wild-type mice.^{141 142} Mice infected with coxsackievirus B3 and fed either L-NMMA or L-NAME had higher viral titers and increased mortality compared with similarly infected animals not given an NOS inhibitor.¹⁴² Experiments with both pharmacological NO donors and with NOS inhibitors have shown that NO_x decreases endothelial cell activation and expression of specific cell-surface adhesion molecules that result in neutrophil and monocyte adhesion and platelet aggregation and also diminishes microvascular permeability.^{27 144 145 146 147 148 149 150 151} NO donors were found to be protective in a heterotopic rat cardiac transplant model after a period of relative hypoxia while the donor heart was maintained in a preservation buffer; this protective effect was presumably due to the ability of NO to quench oxygen-derived free radicals.¹⁵² Finally, "constitutive" expression of NOS2 has been demonstrated in "normal" epithelial layers in vertebrates, including humans, reinforcing the notion that this NOS isoform plays an important role in the initial immune response to many pathogens and in the selective immune response to specific organisms.^{153 154} Thus, within the heart, temporal and spatial restriction of NOS2 activation, as in other tissues, may serve to focus NO_x generation in an appropriate inflammatory response, whereas extensive and unrestricted NOS2 activation may lead to generalized myocardial dysfunction.

Physiological Roles of NOS3 Activation
With the exception of the regulation of coronary blood flow, a discussion of which is beyond the scope of this review, other functional roles of NO_x generated by "constitutively" expressed NOS isoforms, predominantly NOS3 in the microvascular and endocardial endothelium and in cardiac myocytes, have only recently become apparent. It is likely that additional autocrine and paracrine physiological actions of NO_x will be described. In addition, neither the sequence of intracellular signaling events leading to NOS3 activation (particularly in cardiac myocytes) nor the full identities of those downstream signaling cascades regulated by NO_x within cells are yet well understood. Much published evidence to date is inferential, based on the actions of pharmacological NO donors or agents that mimic some actions of cGMP. This section of the review will focus initially on those physiological actions of NOS3 activation in cardiac myocytes thought to be mediated by activation of guanylyl cyclase, followed by evidence of cGMP-dependent paracrine microvascular endothelial control of cardiac muscle function by NO_x. We will conclude with a brief discussion of potential non–GMP-dependent events initiated by exogenous or endogenous sources of NO_x.

Activation of NOS3 by Beating
With the recognition that cardiac myocytes contain a constitutively expressed Ca²⁺/calmodulin-sensitive isoform of NOS, several groups have investigated the role of changes in intracellular Ca²⁺ activity with changes in beating rate on NO_x generation in either isolated papillary muscle or cardiac myocyte preparations. Finkel et al¹⁵⁵ were the first to report that the NOS inhibitor L-NMMA reversed the negative force-frequency relationship ("Bowditch" or "treppe" effect) they observed in hamster papillary muscle preparations. When electrically paced over a range of frequencies from 1 to 5 Hz, L-NMMA led to greater tension development at any given pacing frequency above 1 Hz. This action of L-NMMA could be mimicked by methylene blue, an inhibitor of NOS and guanylyl cyclase, and blunted by the addition of sodium nitroprusside, implicating a causal role of increased endogenous generation of NO_x in the negative inotropic effect of higher pacing frequencies in this model. Kaye et al¹¹² have also shown that activation of NOS3 by higher pacing frequencies in isolated electrically stimulated adult rat ventricular myocytes results in a depression of the positive amplitude of contraction-frequency response in these cells. Activation of NOS3 with pacing was verified by measuring NO_x release into myocyte-conditioned medium, which increased as a function of pacing frequency. Both increased NO_x generation and the depression of the positive rate staircase with pacing could be reversed by the NOS inhibitor L-NA. cGMP-dependent signaling pathways were implicated in both reports for mediating the actions of NO_x with pacing, since 8-bromo-cGMP mimicked the action of sodium nitroprusside on tension development in the isolated paced hamster papillary muscle, whereas the relatively selective guanylyl cyclase inhibitor LY83583 (5 µmol/L) or methylene blue both increased isolated myocyte amplitude of shortening at higher pacing frequencies.^{112 155}

The specific mechanism(s) by which increases in NO_x and cGMP result in a depression in myocyte contractile responsiveness is unknown, although recent data suggest that alterations in myofilament Ca²⁺ sensitivity play a role. Using 8-bromo-cGMP, a lipid-soluble cGMP analogue that is known to be a relatively selective activator of PKG, Shah et al¹⁵⁶ showed that this agent reduced twitch amplitude and steady state diastolic length with no effect on intracellular Ca²⁺ transients in electrically paced adult rat ventricular myocytes loaded with the Ca²⁺-sensitive fluorescent dye indo 1. The effect of 8-bromo-cGMP was inhibited by the protein kinase inhibitor KT5823, at a concentration at which this drug acted as a relatively selective inhibitor of PKG. These authors speculated that PKG phosphorylation of troponin I, which would reduce troponin C's affinity for Ca²⁺, was a possible explanation for these observations. This possibility is supported by preliminary data from our laboratory suggesting that increased activation of NO_x at higher pacing frequencies decreases myofilament Ca²⁺ sensitivity, in part by increasing phosphorylation of troponin I (D.M. Kaye, S.D. Wiviott, X. Han, L. Belhassen, R.A. Kelly, and T.W. Smith, unpublished data, 1996).

Activation of NOS3 by ß-Adrenergic Agonists
In addition to activation of cardiac myocyte NOS3 by increases in time-averaged [Ca²⁺]_i with higher pacing frequencies, it would be expected that NOS3 could also be activated by autonomic agonists that increase [Ca²⁺]_i. The first functional evidence for this was reported by Balligand et al,¹⁰³ who observed that the increased amplitude of shortening of isolated electrically paced adult rat ventricular myocytes in response to the nonselective ß-adrenergic agonist isoproterenol could be enhanced by 30% by removing L-arginine and adding the NOS inhibitor L-NA to the myocyte superfusion medium. This effect of L-NA was observed only at submaximal concentrations of isoproterenol (eg, 2 nmol/L, a concentration that yields an 100% increase in the amplitude of shortening) and not at maximal (eg, 1 µmol/L) concentrations. This observation in isolated myocytes was confirmed in the in situ canine heart by Keaney et al,¹⁵⁷ who used an open-chest model in which the animals underwent bilateral vagotomy, pharmacological autonomic blockade, and placement of a balloon in the aorta to maintain a constant aortic pressure. All hearts were paced at rates above the highest level achieved in response to ß-adrenergic stimulation. An intracoronary infusion of the L-arginine analogue NOS inhibitor L-NAME increased the maximum rate of rise of left ventricular developed pressure (dP/dt_max) in response to intracoronary infusions of either dobutamine or isoproterenol. A significant accentuation of the vasopressor response to racemic dobutamine, a mixed /ß-adrenergic agonist, was observed after the administration of intracoronary L-NAME, as has been reported for NOS inhibitors and other vasoconstrictor agents.¹⁵⁸ However, this effect of L-NAME would have been expected to decrease, not increase, the positively inotropic action of dobutamine in this model. There was no effect of L-NAME on the vasopressor response to isoproterenol.¹⁵⁷ Also, in isolated rat left ventricular papillary muscle, Gomez Llambi et al¹⁵⁹ have shown that an angiotensin II–mediated decline in the isoproterenol-stimulated increase in developed tension in this preparation was mediated by endogenous production of NO_x and could be mimicked in part by 8-bromo-cGMP.

In addition to these in vitro and animal data, Hare et al¹⁶⁰ have reported that intracoronary L-NMMA also ameliorates the increase in left ventricular dP/dt in response to either intracoronary or systemic infusions of dobutamine in human subjects with normal coronary arteries undergoing cardiac catheterization for evaluation of symptomatic left ventricular dysfunction (average ejection fraction, 21%). Heart rate was held constant by right atrial pacing, and dobutamine infusions were targeted to achieve an increase in peak left ventricular dP/dt of 30% to 50%. Intracoronary L-NMMA potentiated the increase in response to dobutamine by 30%. Although these results in humans and experimental animals do not speak to the source of NO_x generated in response to adrenergic agonists in these preparations, the similarity of these data with those generated in isolated paced myocytes suggests that NOS3 activation in cardiac myocytes was responsible. In support of this interpretation, in a preliminary report Kanai et al¹⁶¹ could detect a transient release of NO from either beating neonatal or quiescent adult rat ventricular myocyte preparations in response to the addition of norepinephrine to the culture medium using an NO-selective porphyrinic microsensor.

Finally, not all investigators have found clear evidence for an effect on myocardial contractility of NO_x delivered by pharmacological NO donors or by activation of constitutive NOS. Weyrich et al¹⁶² examined the effect of authentic NO or of NO donors on tension development in isolated rat and rat papillary muscles paced at 1 Hz. Only at a relatively high concentration of norepinephrine (5 µmol/L) was a modest effect of NO observed. These data do not necessarily conflict with those previously cited in this review, since significant effects of NOS inhibition were generally observed only in papillary muscle or isolated myocyte preparations at pacing frequencies above 1 Hz. The disadvantages of NO donors as a source of NO_x are discussed below.

Activation of NOS3 by Muscarinic Cholinergic Agonists
Acetylcholine has long been known to elevate cGMP levels in cardiac muscle, suggesting a link between muscarinic cholinergic signaling and NOS activation in cardiac myocytes.¹⁰³ Muscarinic cholinergic agonists also are well known to exhibit a marked negative inotropic effect in cardiac muscle prestimulated by agents such as ß-adrenergic agonists that increase intracellular cAMP, a phenomenon known as "accentuated antagonism." Since NO was known to activate guanylyl cyclase and might thereby antagonize the actions of agents that increase intracellular cAMP (see above), it was possible that activation of NOS3 and release of NO_x could be one mechanism linking muscarinic cholinergic receptor activation to downstream signaling events. This possibility was supported by the observation of Balligand et al¹⁰³ that the decrease in spontaneous beating rate of confluent primary cultures of neonatal rat ventricular myocytes that could be elicited by the acetylcholine analogue carbachol was reversed by hemoglobin, which binds and inactivates NO_x, or by L-NMMA, a selective NOS inhibitor. NO_x released by carbachol-treated neonatal rat ventricular myocytes could be detected by monitoring the activation of guanylyl cyclase in the RFL-6 rat lung fibroblast cell line, a sensitive bioassay for NO release.¹⁰³

Enlarging on these observations, MacDonell et al¹⁶³ and Han and colleagues^{102 164} have shown subsequently that the accentuated antagonism of ß-adrenergic agonist stimulation of I_Ca-L by the muscarinic cholinergic agonist carbamylcholine in mammalian sinoatrial and atrioventricular nodal cells was dependent on activation of a constitutively expressed NOS in these two cell types. As in ventricular myocytes, this isoform was identified as NOS3 by immunohistochemistry.¹⁰² Preincubation of sinoatrial or atrioventricular nodal cells with a selective L-arginine analogue NOS inhibitor or the addition of methylene blue to the pipette during whole-cell patch-clamp experiments prevented the action of carbamylcholine to suppress ß-adrenergic agonist–enhanced increases in I_Ca-L (Fig 3). Similar data have been reported by Balligand et al⁵⁰ on the regulation of I_Ca-L in whole-cell patch-clamp recordings in isolated adult rat ventricular myocytes.

View larger version (28K): [in this window] [in a new window]   Figure 3. Regulation of ICa-L by acetylcholine (ACh) in the presence of a ß-adrenergic agonist in rabbit sinoatrial nodal cells. In sinoatrial nodal cells, suppression of isoproterenol-stimulated ICa-L by the muscarinic cholinergic agonist ACh appears to be mediated by a type II cGMP-stimulated cAMP phosphodiesterase.102 164 165 AC indicates adenylyl cyclase; MB, methylene blue; ISO, isoproterenol; and LY83583, an inhibitor of guanylyl cyclase. Adapted from Reference 165.

Inhibition of NO-dependent signaling pathways was observed to have little or no effect on I_Ca-L in the experimental preparations described above in the absence of agents that elevate intracellular cAMP, regardless of the presence of carbachol.^{50 102 164 165 166} NOS inhibitors also had no effect on muscarinic cholinergic agonist stimulation of I_K(ACh), which is known to be mediated by ß G-protein subunits, possibly by direct interactions of ß with this ion channel.¹⁶⁷ Also, Zakharov et al¹⁶⁸ failed to demonstrate any effect of NOS inhibitors on muscarinic cholinergic agonist–mediated inhibition of a cAMP-dependent Cl^- current in isolated adult rat ventricular myocytes.

However, in isolated adult feline atrial myocytes, Wang and Lipsius¹⁶⁹ have demonstrated that acetylcholine does lower basal I_Ca-L and amplitude of shortening and that abrupt removal of acetylcholine results in a rebound increase in I_Ca-L and in myocyte amplitude of shortening under "basal" conditions in this myocyte phenotype. Both the initial reduction of I_Ca-L and the rebound in Ca²⁺ current after the removal of acetylcholine were diminished with L-NMMA. Addition of acetylcholine to myocytes pretreated with high concentrations of isoproterenol and forskolin or with the type III PDE inhibitor milrinone resulted in the expected decline in I_Ca-L, but there was no longer a rebound increase in Ca²⁺ current upon removal of acetylcholine.¹⁶⁹ These authors interpreted their data as indicating that adult feline atrial myocytes exhibit a relatively high tonic level of adenylyl cyclase activation that was compensated in part by high endogenous cAMP PDE activity, since milrinone significantly increased "basal" I_Ca-L in these cells. Therefore, the actions of acetylcholine could be explained in part by M₂ cholinergic receptor–mediated activation of NOS and guanylyl cyclase, resulting in an increase in intracellular cGMP. This would result in both activation of a PKG, which would result in a decrease in I_Ca-L (the initial effect of acetylcholine on I_Ca-L could be mimicked by 8-bromo-cGMP), and the inhibition of the type III cGMP-inhibited cAMP PDE. A temporal disparity in the return of these two cGMP-regulated signaling pathways to baseline could account for the rebound in I_Ca-L upon removal of acetylcholine in this experimental model.¹⁶⁹

The relative importance of PKG and of cGMP-regulated PDE's in mediating the signaling events downstream from NOS activation in cardiac myocytes is the subject of a number of published reports and much speculation. MacDonell et al,¹⁶³ for example, could find no evidence that muscarinic agonist–induced increases in cGMP mediated their accentuated antagonism of ß-adrenergic agonists in isolated adult rat ventricular myocytes (paced at 0.5 Hz at 30°C), nor could Mery et al¹⁷⁰ in studies of isolated frog ventricular myocytes. In contrast, Han and colleagues^{102 164 165} provide evidence in adult rabbit sinoatrial and atrioventricular nodal cells that NO-mediated muscarinic cholinergic regulation of I_Ca-L is due in large part to increased activity of the type II cGMP-stimulated cAMP PDE, since neither 8-bromo-cGMP, an activator of PKG, nor milrinone, an inhibitor of type III cGMP-inhibited PDE, affected NO-dependent signaling in these cells. Sumii and Sperelakis¹⁷¹ and Mubagwa et al,¹⁷² studying neonatal rat and adult guinea pig ventricular myocytes, respectively, demonstrated that a PKG in both myocyte preparations clearly acts to suppress I_Ca-L, both in the basal state and particularly after intracellular cAMP levels have been increased. These observations, when combined with the results of experimental protocols using pharmacological NO donors by Wahler and Dollinger¹⁷³ and by Fischmeister and colleagues^{174 175 176} (reviewed in more detail below), suggest that some important differences among species exist not only for specific NO cGMP-dependent signaling pathways (eg, amphibian compared with mammalian ventricular myocytes) but also between specific myocyte phenotypes within a species (eg, atrial or specialized myocyte conduction cells compared with ventricular myocytes). The physiological status of the cellular preparation (eg, beating or paced versus quiescent or resting) may also be important.

A role for an endogenous NO_x-dependent signaling pathway in mediating muscarinic cholinergic signaling has also been determined in an in situ canine heart preparation.¹⁷⁷ After administration of intracoronary dobutamine, electrical stimulation of the vagal nerves resulted in accentuated antagonism of the positively inotropic effect of the adrenergic agonist, which could largely be prevented by concurrent administration of L-NMMA. Selective intracoronary infusions of L-NMMA had no effect if coinfused with L-arginine.¹⁷⁷ Interestingly, intracoronary 8-bromo-cGMP also diminished the expected increase in left ventricular dP/dt, implicating a cGMP-dependent protein kinase in NO-mediated signaling, as in the other mammalian ventricular muscle or isolated myocyte preparations discussed above.

Although the molecular signaling cascades downstream from the activation of NOS3 in cardiac myocytes are coming into better focus, it remains unknown which specific signal transduction pathways link muscarinic cholinergic receptors with NOS activation in these cells. The predominant muscarinic cholinergic receptor in these cells is the M₂ G-protein–coupled receptor. Signaling is initiated by pertussis toxin–inhibited and ß subunit dissociation, leading to inhibition of adenylyl cyclase and acetylcholine-sensitive I_K(ACh), respectively. However, some activation of phospholipase C has also been shown to occur in some cell types expressing these receptors.¹⁷⁸ Indeed, Sterin-Borda et al¹⁷⁹ demonstrated that carbachol stimulates phosphoinositide turnover in adult rat atrial muscle. Pharmacological inhibitors of phospholipase C and PKC mimicked the ability of L-NMMA to induce a rightward shift in the concentration-effect curve of carbachol on the rate of generation of contractile tension (dF/dt) in isolated adult rat atrial muscle stripes stimulated at 150 bpm.¹⁷⁹ This suggests that NOS3 activation by M₂ GRPs may be mediated in part by activation of a phospholipase C in these cells, classically thought to be associated with M₁-, M₃-, and M₅-dependent signaling.¹⁷⁸ The demonstration of some pertussis toxin–insensitive regulation of I_Ca-L in amphibian atrial myocytes and the apparent heterogeneity of muscarinic cholinergic receptors in competitive binding experiments in rat ventricular myocytes indicate that the specific signaling pathways leading to NOS3 activation by acetylcholine in cardiac myocytes remain to be fully elucidated.^{180 181} In addition, Aprigliano et al,¹⁸² in a preliminary report, indicate that accentuated antagonism of ß-adrenergic signaling by muscarinic agonists is ß-adrenergic GRP subtype–selective in neonatal rat ventricular myocytes, suggesting that the effects of the activation of NOS3 in these cells will also depend on the specific adrenergic receptor isoforms that have been activated. Interestingly, another example of NO-dependent coupling of muscarinic cholinergic receptors to a Ca²⁺ current has been demonstrated in neuroblastoma cells.¹⁸³ Finally, coupling of adenosine A₁ receptors to I_Ca-L in isolated rabbit atrioventricular nodal cells has also been shown to be No_x dependent.¹⁸⁴

Activation of NOS3 by Inflammatory Cytokines
A number of inflammatory cytokines have been shown to have rapid (minutes) as well as delayed (hours) effects on the contractile function and in isolated and in situ cardiac preparations (see review by Ungureanu-Longrois et al¹²⁷ ). The delayed negatively inotropic effects of cytokines have been most often attributed to induction of NOS2 in cellular constituents of cardiac muscle, including cardiac myocytes, as reviewed below. Both NO_x-dependent and NO_x-independent rapid effects of specific cytokines have also been described. Finkel et al¹⁸⁵ have described a moderate decline in contractile amplitude in isolated hamster papillary muscles within 2 to 5 minutes of application of relatively high concentrations of recombinant human TNF, IL-2, or IL-6 (but not IL-1) that could be inhibited in each case by L-NMMA. Goldhaber et al¹⁸⁶ have also reported that TNF at high concentrations causes a rapid (10- to 20-minute) decline in peak contractile amplitude of isolated paced adult rabbit ventricular myocytes that could be prevented by L-NAME or by hemoglobin. These authors also noted that this NO_x-dependent decline in twitch amplitude was not accompanied by a decrease in intracellular Ca²⁺ transients, implicating a decrease in myofilament Ca²⁺ sensitivity. These data are in contrast to those of Yokoyama et al¹⁸⁷ in adult feline ventricular myocytes, which showed that lower concentrations of TNF (200 U/mL) led to a rapid decline in both intracellular Ca²⁺ transients and myocyte shortening that was unaffected by myocyte pretreatment with either L-NA or L-NMMA. Similarly, Liu and Schreur¹⁸⁸ have demonstrated a rapid suppression in peak I_Ca-L current by IL-1ß in adult rat ventricular myocytes using a whole-cell patch-clamp technique. Although a role for NO_x was not specifically excluded in this report, the authors noted that intracellular dialysis with a nonhydrolyzable GTP analogue, GTPS, which cannot be metabolized to cGMP, had no effect on IL-1ß–mediated suppression of I_Ca-L. Although these reports have yielded conflicting interpretations, it appears that relatively high concentrations of some cytokines may induce an NO_x-dependent rapid decline in inotropic state that precedes the expression of NOS2. The physiological relevance of these observations has yet to be determined.

Paracrine Actions of Endothelium-Derived NO_x on Cardiac Function
Clear evidence for the regulation of cardiac muscle function by NO_x generated by coronary vascular and endocardial endothelium has also been reported. Indeed, the first report of any effect of NO_x (initially identified as endothelium-derived relaxing factor) on myocardial signal transduction was that of Smith et al,¹⁸⁹ who demonstrated that substance P elevated cGMP levels in isolated ferret papillary muscles. This effect of substance P could be inhibited either by hemoglobin or by selective removal of the overlying endocardial endothelium. This laboratory reported subsequently that both bradykinin and substance P, assumed to be relatively endothelium-selective NO-dependent vasodilators, induced characteristic changes in left ventricular function in an isolated guinea pig working (ejecting) heart.¹⁹⁰ Both agonists accelerated early left ventricular relaxation in diastole with only a minimal negative inotropic effect. The effects of both substance P and bradykinin were inhibited by the addition of hemoglobin to the perfusion buffer. In a separate report, Grocott-Mason et al¹⁹¹ also noted similar pharmacodynamic actions of sodium nitroprusside on diastolic relaxation in the same experimental preparation.

Using isolated guinea pig ventricular myocytes in short-term coculture with confluent bovine aortic endothelial cells, Brady et al¹⁹² demonstrated that bradykinin significantly reduced myocyte amplitude of shortening when administered in coculture but not when applied to the myocytes in the absence of endothelial cells. This effect of bradykinin in coculture could be inhibited by L-NAME. Finally, Paulus et al¹⁹³ have shown that intracoronary infusion of substance P both into human subjects with normal left ventricular function and coronary anatomy and into cardiac transplant recipients causes a modest decline in systolic performance and increased end-diastolic distensibility. These hemodynamic effects were not reproduced when substance P was infused into the systemic circulation through the right atrium. Although conclusive evidence was not obtained for a role of local paracrine generation of NO_x, these changes in left ventricular function were qualitatively similar to results obtained in normal subjects by these same authors during selective intracoronary but not right atrial infusions of sodium nitroprusside.¹⁹⁴

Pharmacological NO Donors and Regulation of Cardiac Myocyte I_Ca-L
Studies of the molecular pharmacology of NO_x-dependent signaling with NO-donating drugs, in cardiac myocytes or other cell types, have a number of disadvantages compared with endogenous generation of NO. The kinetics and extent of release of NO and its metabolites from specific NO donors, as well as their charge, stability, and lipophilicity, clearly differ among these agents, from tissue to tissue with the same agent, and in the same tissue depending on cellular redox state; other factors are also involved.^{195 196 197} Some NO donors or their metabolites have been demonstrated to have effects not directly related to NO generation (eg, the generation of oxygen radicals by SIN-1). Finally, NO donors cannot mimic the temporal and spatial characteristics of NO_x released by endogenous NOS and may induce NO-dependent effects that are irrelevant to physiological NO_x-dependent signaling under many conditions. Nevertheless, with these limitations in mind, these agents have proven useful in generating and testing hypotheses and in elucidating components of specific NO-dependent signaling pathways. A more general discussion of this pharmacology is beyond the scope of this review (see Feelisch and Stamler¹⁹⁶ for a detailed review). We will focus briefly on the use of these agents to examine the possible mechanisms by which endogenously generated NO could regulate I_Ca-L.

cGMP-Mediated Effects of NO Donors
Mery et al¹⁷⁵ originally reported that SIN-1, the active metabolite of molsidomine, has a biphasic effect on I_Ca-L in enzymatically dissociated frog ventricular myocytes. Although the NO donor had little effect on basal I_Ca-L, low concentrations of SIN-1 (0.1 to 10 nmol/L) were found to induce a moderate stimulation of I_Ca-L, whereas higher concentrations (>100 nmol/L) markedly suppressed I_Ca-L after stimulation of adenylyl cyclase with either forskolin or isoproterenol. Since this initial stimulation of I_Ca-L by SIN-1 could be inhibited by milrinone, whereas SIN-1 suppression of forskolin activation of I_Ca-L could be inhibited by intracellular dialysis with nonhydrolyzable 8-bromo-cAMP, these authors interpreted their data as indicating that these two actions of SIN-1 were mediated by a type III cGMP-inhibited cAMP PDE and a type II cGMP-stimulated cAMP, respectively.¹⁷⁵ Kirstein et al¹⁷⁶ reported subsequently that low concentrations of SIN-1 enhanced basal I_Ca-L in primary isolates of adult human atrial cells isolated from specimens obtained during open-heart surgery. This effect of SIN-1 was mimicked by, but not additive to, that of milrinone, implying that basal adenylyl cyclase activity was relatively high in these cells and that, under these conditions, myocyte cAMP levels and I_Ca-L were tonically suppressed by a type III cGMP-inhibited cAMP PDE. As in frog myocytes, concentrations of SIN-1 higher than 100 µmol/L tended to decrease I_Ca-L.¹⁷⁶

In contrast, in an earlier study, Mery et al¹⁷⁴ had reported that suppression of I_Ca-L in adult rat ventricular myocytes that had been pretreated with the nonselective PDE inhibitor IBMX or a nonhydrolyzable cAMP analogue was likely due to activation of PKG and not to cGMP-regulated PDEs. Wahler and Dollinger¹⁷³ reported a similar conclusion using SIN-1 in adult guinea pig ventricular myocytes. SIN-1 had little effect on basal I_Ca-L, whereas low concentrations caused a modest stimulation of I_Ca-L with submaximal (10 nmol/L) concentrations of isoproterenol. A high concentration of SIN-1 (100 µmol/L) consistently inhibited I_Ca-L prestimulated by IBMX or isoproterenol or by the nonhydrolyzable cAMP analogue 8-bromo-cAMP.¹⁷³ However, SIN-1's inhibitory effect on I_Ca-L was prevented by either LY83583 or by KT5823, relatively selective inhibitors of guanylyl cyclase and of PKG, respectively. Similarly, Kojda et al¹⁹⁸ observed that low concentrations of the NO donors SNAP and DEA/NO, as well as several organic nitrates, caused a moderate positively inotropic effect in adult rat ventricular myocytes that could be prevented by Rp-cAMPS, an agent that is known to inhibit cAMP-dependent protein kinases. Higher concentrations of either SNAP or DEA/NO (100 µmol/L) suppressed myocyte contractile function, a decrease that could be reversed by addition of the relatively selective PKG inhibitor KT5823. Mohan et al¹⁹⁹ have reported a similar biphasic inotropic response to NO donors in isolated feline papillary muscle strips that appeared to be dependent on a cGMP-mediated signaling pathway. Taken together with data reviewed above regarding muscarinic cholinergic control of ß-adrenergic signaling in cardiac myocytes, these reports emphasize again the differences among distinct myocardial cell phenotypes (eg, atrial versus ventricular myocytes) within closely related species.

Non-cGMP–Mediated Effects of NO Donors
Although an extensive discussion of non-cGMP–mediated actions of NO_x is beyond the scope of this review, there are now data implicating additional physiological and pathophysiological mechanisms by which NO_x could affect cardiac function that deserve mention in this context. NO_x is known to bind covalently to, and alter the function of, a number of cellular proteins, including enzymes and transcriptional regulatory factors, among others. This can occur by direct or N₂O₃-mediated S-nitrosylation, by the intermediate formation of metal NO adducts, in combination with superoxide anion (O₂^-), by peroxynitrite, and by the formation of nitrotyrosines.^{3 200 201 202} The actions of NO_x within a cell, therefore, would depend on their concentration, the cellular redox state, the abundance of metals, protein thiols, and low-molecular-weight thiols (such as glutathione), as well as other nucleophile targets. Many nitrosation reactions are reversible and could serve a physiological regulatory role. The description by Chiamvimonvat et al²⁰³ of reversible inhibition of I_Ca-L by sulfhydryl-modifying agents in CHO cells stably transfected with pore-forming subunits of the rabbit L-type Ca²⁺ channel suggests that direct S-nitrosylation of these thiols by endogenously generated NO_x or by pharmacological NO donors could similarly affect channel function.

Some of the most intriguing data to date involve the effects of NO_x on myocardial energetics. NO_x has been shown in a number of tissues to inhibit mitochondrial respiration and enzymes involved in glycolysis. It has been suggested that relevant mechanisms may include S-nitrosylation of thiols, formation of nitrotyrosines, NO_x binding to iron sulfur clusters, or NO_x binding to heme-containing proteins in the mitochondrial respiratory chain.^{200 201 202 203 204 205 206 207 208 209 210 211 212} NO_x has also been shown to inactivate glyceraldehyde-3-phosphate dehydrogenase by covalent linkage to NAD or NADH.^{208 209} Irrespective of the specific mechanism(s), several groups have reported physiologically relevant effects of both pharmacological NO donors and endogenous sources of NO_x on energetics in striated muscle.^{28 213 214} In two reports, Shen and colleagues^{213 214} demonstrated that systemic administration of an L-arginine analogue NOS inhibitor to dogs resulted in both an increase in systemic vascular resistance and an increase in oxygen consumption, both in whole animal and in isolated hindlimb preparations. The changes in oxygen consumption were not mimicked by an intravenous infusion of methoxamine, a vasoconstrictor that caused an increase in systemic vascular resistance comparable to that of the NOS inhibitor.²¹³ Addition of either a NO donor or an endogenous NO_x-dependent vasodilator such as bradykinin to isolated skeletal muscle slice preparations in vitro decreased oxygen consumption by these tissue samples.²¹⁴ Indeed, both the NO donor and bradykinin could lower oxygen consumption of skeletal muscle slices even in the presence of a mitochondrial H⁺ ionophore, which uncouples oxygen consumption from oxidative phosphorylation. These authors concluded that their data implicate direct inhibition of the mitochondrial electron transport chain by NO_x. Oddis and Finkel²¹⁵ have recently shown that IL-1ß induced NOS2 expression in neonatal rat ventricular myocytes, decreasing mitochondrial respiration in these cells. This decrease could be reversed by adding L-NMMA to the medium. One likely target of NO_x is the heme moieties in the cytochrome chain, particularly cytochrome C oxidase.^{204 205 210} Finally, Bates et al²¹⁶ have reported localization of NOS3 within mitochondria using immunoglobal labeling and electron microscopy isolated from adult rat ventricular muscle and other tissues. Although intriguing, these results require confirmation before any discussion of their significance.

A number of laboratories have now investigated the potential role of endogenous NO_x generation in the regulation of myocardial energetics. In preliminary reports, two groups have examined the relationship between myocardial oxygen consumption in the presence and absence of an NOS inhibitor in dog hearts in situ when workload was varied either by infusion of isoproterenol or by increasing heart rate by pacing.^{217 218} In both cases, the NOS inhibitor decreased oxygen consumption at any given workload, apparently increasing the efficiency of oxygen utilization, although these preliminary reports may have been confounded by systemic effects of NOS inhibition. An increase in workload was required to manifest this effect of endogenously produced NO_x, since minimal, if any, effects of NO_x on energetics in the heart have been detected under resting or basal conditions.^{219 220}

Among the potential cellular mechanisms that could contribute to these findings, our laboratory has shown recently that infusion of the pharmacological NO donor SNAC into isolated retrogradely perfused adult rat hearts diminishes cardiac contractile responsiveness to an inotropic stimulus (a step increase in Ca²⁺ in the perfusion buffer).²²¹ SNAC-treated hearts exhibited an immediate decline in ATP content, as measured by ³¹P nuclear magnetic resonance spectroscopy but only a modest decline in phosphocreatine content, in contrast to hearts exposed to high Ca²⁺ buffer without SNAC, in which the expected and opposite response occurred (ie, no change in ATP and an abrupt fall in phosphocreatine). Among other possible causes, these data suggested that phosphoryl transfer by creatine kinase was being inhibited by the NO donor. In addition, the enzymatic activity of purified creatine kinase enzyme in solution could be inhibited by several NO donors. This inhibition could be rapidly reversed by sulfhydryl reducing agents such as dithiothreitol.²²¹

Thus, NO_x generated under physiological conditions by endothelial and/or myocyte NOS3 activity or NO_x generated under pathophysiological conditions by vascular or myocyte NOS2 could affect myocardial energetics by one of several mechanisms. Interestingly, Boekstegers et al²²² have recently reported that exposure of neonatal rat ventricular myocytes for 24 hours to TNF resulted in a reversible decline in their inotropic responsiveness to isoproterenol or to increased extracellular Ca²⁺. These changes were accompanied by alterations in high-energy phosphate concentrations that paralleled those we observed in the intact rat heart in response to SNAC (ie, a decline in ATP with little or no change in phosphocreatine). These authors did not address the role of endogenous generation of NO_x in their experiments, however. In any event, the role of NO_x in the regulation of cardiac energetics must remain speculative until the studies reviewed above can be confirmed and extended.

The explosion of reports on NO to which we refer in the opening paragraph has generated much light as well as smoke and heat. As in the case of vascular physiology and biology, the discovery and characterization of myocardial NO_x-dependent signaling pathways have clarified numerous aspects of specific signal transduction cascades in cardiac muscle, while generating additional apparent paradoxes and new hypotheses to be tested. It is likely to remain a rapidly growing but maturing field in cardiovascular research.

	Selected Abbreviations and Acronyms

 

AP = activation protein CAT = cationic amino acid transporter DEA/NO = 2,2-diethyl-L-hydroxy-L-nitroso-hydrazine DIG = detergent-insoluble glycosphingolipid-enriched complex eNOS, NOS3 = endothelial constitutive NOS GAS = gamma activating sequence H4B = tetrahydrobiopterin IBMX = isobutylmethylxanthine ICa-L = L-type voltage-gated Ca2+ current IFN = interferon gamma IK(ACh) = acetylcholine-sensitive K+ current IL = interleukin iNOS, NOS2 = cytokine-inducible NOS L-NA = nitro-L-arginine L-NAME = NG-nitro-L-arginine methyl ester L-NMMA = NG-monomethyl-L-arginine LPS = lipopolysaccharide MAPK = mitogen-activated protein kinase NF = nuclear factor nNOS, NOS1 = neuronal NOS NOS = NO synthase NOx = nitrogen oxides PDE = phosphodiesterase PKA, PKC, PKG = protein kinases A, C, and G PSD = postsynaptic density RT-PCR = reverse-transcription polymerase chain reaction SIN-1 = 3-morpholinosydnonimine SNAC = S-nitrosoacetylcysteine SNAP = S-nitroso-N-acetyl-D,L-penicillamine STAT = signal transducers and activators of transcription TGF = transforming growth factor TNF = tumor necrosis factor

	Acknowledgments

 
This study was supported by grants from the National Institutes of Health (HL-36141 and HL-52320). We thank Thomas Michel and Xinqiang Han for their careful reading of the manuscript and for many helpful discussions.

	Footnotes

 
This manuscript was sent to Leslie A. Leinwand, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received February 9, 1996; accepted May 22, 1996.

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