Cardiovascular Research

Cardiovascular Research 2004 61(3):402-413; doi:10.1016/j.cardiores.2003.09.019
© 2004 by European Society of Cardiology

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Copyright © 2003, European Society of Cardiology

Nitric oxide in myocardial ischemia/reperfusion injury

Rainer Schulz^*,a, Malte Kelm^b and Gerd Heusch^a

^aInstitut für Pathophysiologie, Zentrum für Innere Medizin, Universitätsklinikum Essen, Hufelandstr. 55, 45122 Essen, Germany
^bKlinik für Kardiologie, Pneumologie und Angiologie, Universitätsklinikum der Heinrich-Heine Universität Düsseldorf, Düsseldorf, Germany

^* Corresponding author. Tel.: +49-201-7234480; fax: +49-201-7234481. rainer_schulz{at}uni-essen.de

Received 8 July 2003; revised 29 August 2003; accepted 15 September 2003

	Abstract

Top Abstract 1. Introduction 2. Production of nitric... 3. Metabolism of nitric... 4. NO-cyclic guanosine... 5. Coronary vascular effects... 6. Inflammation and NO 7. Cardiomyocyte function 8. Cardiomyocyte mitochondria... 9. NO and apoptosis 10. Considerations on... 11. Importance of NO... 12. Importance of NO... 13. Final conclusion References

 
Administration of nitric oxide (NO), NO donors or drugs that enhance NO realease (statins, calcium antagonists, ACE-inhibitors, dexamethasone) prior to ischemia protects the myocardium against ischemia/reperfusion injury. While this exogenous administration of NO prior to ischemia can initiate a preconditioning-like phenomenon, endogenous NO-synthase (NOS)-derived NO is not involved in triggering or mediating the early phase of ischemic preconditioning's protection, but does play a pivotal role for initiating and mediating the delayed phase of ischemic preconditioning's protection.

The present review now summarizes the importance of endogenous and exogenous NO when given at the time of reperfusion for vascular and myocardial function and morphological outcome following ischemia/reperfusion. Given the inconsistency of the published data, potential confounding factors that might affect experimental results on the role of NO in myocardial ischemia/reperfusion were identified, such as (1) the lack of characterization of the involved NOS isoforms in myocardial ischemia/reperfusion injury in different animal species, (2) the lack of direct measurements of myocardial NO concentration and/or NOS activity to assure sufficient NOS inhibition, (3) the lack of consideration of nonenzymatic NO production as a potential source of NO, and (4) the absence of plasma or blood components in in vitro studies influencing NO delivery and metabolism.

Future research on the importance of NO in ischemia/reperfusion injury will have to focus more precisely on the identification and standardization of potential confounding experimental factors that influence synthesis, transport, and interaction of NO with various targets in blood and tissue.

KEYWORDS Nitric oxide; Infarct size; Stunning

	1. Introduction

Top Abstract 1. Introduction 2. Production of nitric... 3. Metabolism of nitric... 4. NO-cyclic guanosine... 5. Coronary vascular effects... 6. Inflammation and NO 7. Cardiomyocyte function 8. Cardiomyocyte mitochondria... 9. NO and apoptosis 10. Considerations on... 11. Importance of NO... 12. Importance of NO... 13. Final conclusion References

 
Administration of nitric oxide (NO) or NO donors prior to ischemia attenuates the consequences of myocardial ischemia/reperfusion; i.e., reduces infarct size and endothelial dysfunction [1]. These beneficial effects of NO are related to a pharmacological type of preconditioning, and the existing literature has been reviewed extensively by Roberto Bolli [1]. Also, pretreatment with drugs that enhance NO release such as statins [2], certain calcium antagonists [3], ACE inhibitors [4] or dexamethasone [5] protects the myocardium against ischemia/reperfusion injury. While exogenous administration of NO prior to ischemia can initiate a preconditioning-like phenomenon, endogenous NO-synthase derived NO is not involved in triggering or mediating the early phase of ischemic preconditioning's protection (for review, see Ref. [6]), but does play a pivotal role for initiating and mediating the delayed phase or second window of ischemic preconditioning's protection (for review, see Ref. [7]). To what extent NO is involved in the pharmacologically induced second window of protection is still a matter of debate (for review, see Ref. [8]).

Given the above established facts, the present review will concentrate (1) on the importance of endogenous NO for vascular and myocardial function in myocardial ischemia/reperfusion, and (2) on the importance of exogenous NO when given at the time of reperfusion for the functional and morphological outcome following myocardial ischemia/reperfusion, and (3) to identify potential confounding factors that might affect experimental results on the role of NO in myocardial ischemia/reperfusion and which could explain some of the inconsistency of the results obtained so far.

	2. Production of nitric oxide

Top Abstract 1. Introduction 2. Production of nitric... 3. Metabolism of nitric... 4. NO-cyclic guanosine... 5. Coronary vascular effects... 6. Inflammation and NO 7. Cardiomyocyte function 8. Cardiomyocyte mitochondria... 9. NO and apoptosis 10. Considerations on... 11. Importance of NO... 12. Importance of NO... 13. Final conclusion References

 
Nitric oxide synthases (NOS) are the enzymes responsible for NO generation. To date, three distinct NOS isoforms have been identified: neuronal NOS (type 1), inducible NOS (type 2) and endothelial NOS (type 3). NOS's catalyze an overall 5-electron oxidation of one N-atom of the guanidino group of L-arginine to form NO and L-citrulline, with the intermediate N-hydroxy-L-arginine (NOHA). NO synthesis is critically influenced by various cofactors such as tetrahydrobiopterin, flavin mononucleotide and flavin adenine dinucleotide, the presence of reduced thiols, the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) and, of course, substrate availability (Fig. 1). Also, NOS1 and NOS3 are dependent on calmodulin and calcium [9]. Without an adequate delivery of substrate and co-factors, NOS no longer produces NO but instead transfers the free electrons to oxygen and thus produces free oxygen radicals [10].

View larger version (26K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Factors contributing to NO production and downstream targets of NO. For details, see text. PKA: protein kinase A, PKB: protein kinase B, PKC: protein kinase C, PI3K: phosphoinositol-3 kinase, CaM: calmodulin, BH4: tetrahydrobiopterin, HSP90: heat shock protein 90, XO: xanthine oxidase, LDH: lactate dehydrogenase, PDE: phosphodiesterase, PK: protein kinase.

 
Under resting conditions, NO synthesis has been mainly attributed to the vascular endothelium and its constitutively active NOS3. Both NOS1 and NOS3 have been identified in cardiomyocytes, and their expression appears to be species-dependent in that NOS1 is more important in rats while NOS3 is of greater importance in rabbits [11]. The expression of NOS differs within the left ventricular wall of ferrets and humans, with a higher expression in the subepicardium than in the subendocardium [12], and also the subcellular distribution of NOS 1 and NOS 3 within cardiomyocytes differs, with NOS3 present in caveolae located at the outer membrane and NOS1 located at the sarcoplasmic reticulum [13]. Given its distinct location, NOS3 has been proposed to mainly interact with β-adrenoceptors and L-type calcium channels, thereby attenuating calcium influx into the cardiomyocyte [14]. Conversely, NOS1 has been proposed to interact with ryanodine receptors, thereby increasing calcium release from the sarcoplasmic reticulum [15]. Platelets and leukocytes also carry NOS isoforms [16,17]; however, those NOS isoforms contribute significantly to NO formation only upon activation [18,19].

The NOS3 activity is increased by phosphorylation of its serine residue 1177; this phosphorylation is achieved by activation of PI3-kinase and protein kinase B (Akt). In contrast, phosphorylation at the threonine residue 495 by AMP-activated kinase or protein kinase C can inactivate NOS3 [13] (Fig. 1).

The cardiac interstitial NO concentration is within the nanomolar range during normoperfusion [20]. During ischemia, NOS3 activity is increased within minutes [21], and subsequently the NO concentration during early ischemia is increased [22]. However, with prolonged myocardial ischemia, NOS3 protein expression decreases [23,24], and the increased tissue acidosis attenuates NOS3 activity[24]. Within the early seconds of reperfusion, the NO concentration is again increased [23]; however, with prolongation of reperfusion NOS activity and thus NO concentration decrease below baseline values [25]. Also, neurohumoral factors affect NO availability. Angiotensin II, which is increased in concentration during myocardial ischemia [26], is capable of increasing NOS3 protein expression; however, since—at the same time—free radical production is increased, the myocardial NO concentration is decreased [27]. Tumor necrosis factor (TNF), which is rapidly released within the ischemic myocardium [28–30], decreases NOS3 protein expression by reducing eNOS mRNA stability [18], but induces NOS2 protein expression in leukocytes, however not in cardiomyocytes [31].

Apart from NOS-derived NO, a non-enzymatic production of nitric oxide exists during ischemia [32]. Tissue acidosis occurring during ischemia increases NO production independent from NOS3 [33], and even at normal pH, xanthine oxidase in the presence of low pO₂ and high NADH concentration is capable of producing NO from nitrite [34]; the nitrite concentration in plasma amounts to 0.5 µM and in vascular tissue up to 10 µM [35]. Indeed, in isolated rat heart [22] and in rabbit hindlimb muscle [36], the NO concentration is still increased during ischemia after complete NOS inhibition by L-NNA.

In conclusion, the cardiac interstitial NO concentration during early ischemia and early reperfusion is increased. The increase in the NO concentration is in part derived from activated NOS isoforms (species-dependent differences) but also from NOS-independent pathways.

	3. Metabolism of nitric oxide in the mammalian circulation

Top Abstract 1. Introduction 2. Production of nitric... 3. Metabolism of nitric... 4. NO-cyclic guanosine... 5. Coronary vascular effects... 6. Inflammation and NO 7. Cardiomyocyte function 8. Cardiomyocyte mitochondria... 9. NO and apoptosis 10. Considerations on... 11. Importance of NO... 12. Importance of NO... 13. Final conclusion References

 
The charge neutrality of NO facilitates its free diffusion in aqueous solutions and across cell membranes. The biological effects of NO are dependent on its half-life, which depends on the rate of NO formation (see above) and its metabolism. In principle, NO can react by electron gain to form the nitroxyl anion (NO^–) and by electron loss to form the nitrosonium ion (NO⁺). Various metabolic routes and reactions contribute to the breakdown and/or conversion of NO, NO^– and NO⁺, e.g., heme proteins such as guanylyl cyclase, catalase, xanthine oxidase, superoxide dismutase (SOD) and hemoglobin, or high-energy free radicals such as the hydroxyl radical or carbon, oxygen- and nitrogen-centered radicals [10].

The major immediate breakdown product of NO in plasma is nitrite (NO₂^–) [9]. Nitrite can be taken up by red blood cells, where it is oxidized in a hemoglobin-dependent manner to nitrate (NO₃^–), which can subsequently be redistributed into plasma. NO also rapidly interacts with superoxide anions to produce the potent oxidant peroxynitrite (ONOO^–). The formation of free oxygen radicals is increased during reperfusion, depending on duration and severity of the preceding ischemia; thus with more severe and prolonged ischemia free radical formation during the subsequent reperfusion is increased [37], subsequently also increasing the formation of peroxynitrite. High concentrations of peroxynitrite are thought to oxidize thiols or thioethers, to nitrate tyrosine residues, to nitrate and oxidize guanosine, to degrade carbohydrates, to initiate lipid peroxidation, and to cleave DNA. The peroxynitrite in excess decomposes to yield NO₃^–.

Alternatively, NO can react with O₂ to yield reactive intermediates. It is well appreciated that the autooxidation of NO in an aqueous environment leads to the formation of reactive NO species such as dinitrogen trioxide (N₂O₃). This intermediate can nitrosate as well as oxidize different substrates to yield either N-nitroso or S-nitroso compounds. Recent data provide unequivocal evidence for nitrosative chemistry of NO in human plasma. Redox-active thiols, which are abundantly present in plasma, can incorporate NO and transport it throughout the mammalian circulation in the form of bioactive nitrosothiols [38].

In conclusion, depending on its environment (buffer, plasma, blood, high pO₂, presence of free oxygen radicals) NO forms different reactive intermediates which dose-dependently react with surrounding tissue components.

	4. NO-cyclic guanosine monophosphate (cGMP) pathway

Top Abstract 1. Introduction 2. Production of nitric... 3. Metabolism of nitric... 4. NO-cyclic guanosine... 5. Coronary vascular effects... 6. Inflammation and NO 7. Cardiomyocyte function 8. Cardiomyocyte mitochondria... 9. NO and apoptosis 10. Considerations on... 11. Importance of NO... 12. Importance of NO... 13. Final conclusion References

 
The major target of NO in the cardiovascular system is the NO-sensitive guanylyl cyclase or soluble guanylyl cyclase (for review, see Ref. [39]) (Fig. 1). Activation of the guanylyl cyclase results in the conversion of guanosine triphosphate to the second messenger cGMP. CGMP activates two specific cGMP-dependent protein kinases (PKG I and II), PKG I being most important for vasodilation and inhibition of platelet aggregation (for review, see Ref. [40]). CGMP also inhibits the activity of phosphodiesterases (PDE II and III); inhibition of PDE III elevates the concentration of cyclic adenosine monophosphate (cAMP), thereby subsequently increasing the activity of protein kinase A (PKA; for review, see Ref. [41]). A low concentration of cGMP appears to primarily inhibit PDE III, while a higher cGMP concentration activates PKG [39–41].

During ischemia, the ensuing acidosis reduces the guanylyl cyclase activity in isolated rat cardiomyocytes [42], thereby potentially counterbalancing the increased NO concentration during early ischemia. Indeed, the myocardial cGMP concentration did not increase significantly during 40 min of low flow ischemia in isolated buffer-perfused rat hearts [43] and 90 min low flow ischemia in in situ pig hearts [44]. In contrast, the cGMP concentration was increased in ischemic areas compared to normoperfused areas of patients with coronary artery disease [45,46]. Following ischemia/reperfusion, however, myocardial cGMP concentration was decreased compared to baseline in isolated rat hearts as well as in pig hearts in vivo [47].

In conclusion, the cardiac cGMP concentration during ischemia is possibly somewhat increased, while upon reperfusion it is clearly decreased. This time course of the alteration in cGMP concentration matches that of the cardiac NO concentration. Dose-dependently, cGMP inhibits PDE or activates PKG, thereby mediating its effects on the vasculature, platelets and myocytes.

	5. Coronary vascular effects of NO

Top Abstract 1. Introduction 2. Production of nitric... 3. Metabolism of nitric... 4. NO-cyclic guanosine... 5. Coronary vascular effects... 6. Inflammation and NO 7. Cardiomyocyte function 8. Cardiomyocyte mitochondria... 9. NO and apoptosis 10. Considerations on... 11. Importance of NO... 12. Importance of NO... 13. Final conclusion References

 
Pharmacological inhibition of endogenous NO synthesis increases blood pressure [44,48,49], and lack of NOS3 causes mild hypertension in mice [50]. Also in the presence of a coronary stenosis, NO contributes to the maintenance of regional myocardial blood flow [51]. The NO-induced vasodilation results from a decreased intracellular calcium concentration in vascular smooth muscle cells secondary to direct activation of calcium-dependent potassium channels, cGMP-dependent activation of PKGI and cGMP-dependent inhibition of voltage-gated calcium channels (for review, see Ref. [40]). Apart from its direct vasodilatory effect, NO can preserve myocardial perfusion by inhibiting platelet aggregation and leukocyte adherence to the vascular endothelium (Fig. 2), the latter most likely independently from cGMP. NOS activity is reduced during reperfusion [25], as evidenced also by loss of NO-dependent vasodilation in response to acetylcholine [52] or bradykinin [53] or loss of vasoconstriction in response to NOS inhibition [54], and neutrophil adherence to the endothelium progressively increases during reperfusion [55]. Preservation of endothelial function by administration of L-arginine [56], a NO donor [57], or low concentrations of peroxynitrite (1–2 µM) [58] inhibits such neutrophil adherence and their subsequent infiltration following ischemia/reperfusion. Following ischemia/reperfusion, myocardial infiltration of mononuclear cells contributes to irreversible tissue injury, since pharmacological blockade of NOS2—which is located in the mononuclear cells but not in cardiomyocytes—reduced infarct size following 30 min coronary artery occlusion and 48 h reperfusion in rabbits [59].

View larger version (29K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Effects of low (eNOS- or nNOS-derived) or high NO (iNOS-derived) concentrations. VSMC: vascular smooth muscle cells, MPTP: mitochondrial permeability transition pore.

 
Endogenous NO also attenuates the ischemia-induced increase in adenosine. Following NOS inhibition with L-NAME, the myocardial adenosine concentration during ischemia is increased in isolated rabbit hearts [60]. The effect of NOS inhibition on the myocardial adenosine concentration is mediated via protein kinase C and subsequently 5' nucleotidase [61,62].

	6. Inflammation and NO

Top Abstract 1. Introduction 2. Production of nitric... 3. Metabolism of nitric... 4. NO-cyclic guanosine... 5. Coronary vascular effects... 6. Inflammation and NO 7. Cardiomyocyte function 8. Cardiomyocyte mitochondria... 9. NO and apoptosis 10. Considerations on... 11. Importance of NO... 12. Importance of NO... 13. Final conclusion References

 
Both adenosine and TNF are involved in ischemia/reperfusion injury [6,63]. Endogenous NO either directly or through an altered myocardial adenosine concentration [64] facilitates the ischemia-induced increase in the myocardial TNF concentration; accordingly, NOS inhibition completely abolishes the increase in the myocardial TNF concentration secondary to coronary microembolization in dogs [65].

	7. Cardiomyocyte function

Top Abstract 1. Introduction 2. Production of nitric... 3. Metabolism of nitric... 4. NO-cyclic guanosine... 5. Coronary vascular effects... 6. Inflammation and NO 7. Cardiomyocyte function 8. Cardiomyocyte mitochondria... 9. NO and apoptosis 10. Considerations on... 11. Importance of NO... 12. Importance of NO... 13. Final conclusion References

 
As mentioned above, NO via cGMP dose-dependently inhibits PDE and/or activates PKG. At low NO/cGMP concentrations (in nM range), inhibition of PDEIII activity or direct activation of adenylyl cyclase [66] with subsequently increased cAMP concentration and PKA activity increases cardiomyocyte function (for review, see Ref. [41]). Additional mechanisms by which low NO/cGMP concentrations might increase cardiomyocyte function relate to a direct activation of ryanodine receptors or voltage-operated calcium channels [13]. Indeed, pharmacological blockade of NOS3 in pigs in vivo reduced regional myocardial function for a given blood flow and oxygen consumption, supporting a positive inotropic effect of the basal endogenous NO concentration [44]. Loss of NO release following ischemia/reperfusion (see above) could therefore contribute to the loss of postischemic contractile function.

At higher NO/cGMP concentrations (in µM range), activation of PKG inhibits voltage-dependent calcium channels [13,41] and decreases myofilament calcium responsiveness by phosphorylation of troponin I [67]. While a higher NO/cGMP concentration also suppresses the increase of regional myocardial function during β-adrenergic stimulation [68], most likely by directly inhibiting ryanodine receptors, pharmacological blockade of endogenous NOS-dependent NO synthesis in pigs did not impact on β-adrenergic responsiveness [69]. Thus, only at high concentrations might NO/cGMP directly reduce cardiomyocyte function.

	8. Cardiomyocyte mitochondria and NO

Top Abstract 1. Introduction 2. Production of nitric... 3. Metabolism of nitric... 4. NO-cyclic guanosine... 5. Coronary vascular effects... 6. Inflammation and NO 7. Cardiomyocyte function 8. Cardiomyocyte mitochondria... 9. NO and apoptosis 10. Considerations on... 11. Importance of NO... 12. Importance of NO... 13. Final conclusion References

 
Endogenous NO interacts with mitochondrial respiration, possibly at several steps of the electron transfer. In isolated rat hearts subjected to 20 min ischemia and 20 min reperfusion, myocardial ATP content was increased following NOS inhibition, and the production of free oxygen radicals was reduced [70]. Furthermore, in isolated mitochondria, exogenous NO (4 µM) when administered together with calcium increased the production of peroxynitrite in the presence of an unaltered respiration, supporting a shift from oxygen usage for ATP production towards the production of free oxygen radicals [71].

A low concentration of the NO donor SNAP (2 µM) increases the mitochondrial membrane potential via activation of mitochondrial ATP-dependent potassium channels [72]. Any increase in the mitochondrial membrane potential will decrease the mitochondrial calcium uptake, and indeed exogenous NO reduced mitochondrial calcium overload during simulated ischemia [73].

	9. NO and apoptosis

Top Abstract 1. Introduction 2. Production of nitric... 3. Metabolism of nitric... 4. NO-cyclic guanosine... 5. Coronary vascular effects... 6. Inflammation and NO 7. Cardiomyocyte function 8. Cardiomyocyte mitochondria... 9. NO and apoptosis 10. Considerations on... 11. Importance of NO... 12. Importance of NO... 13. Final conclusion References

 
In isolated cardiomyocytes and hearts, high NO concentrations (µM range) induce necrosis and apoptosis [74,75]. The amount of necrosis and apoptosis critically depends on the energetic situation of the cardiomyocytes, with apoptosis favored at preserved ATP pools [76]. While the development of necrosis following NO application appears to be independent of cGMP, the development of apoptosis involves cGMP and subsequently activation of mitogen-activated protein kinases and transcription factors [74,77,78]. Most interestingly, the development of apoptosis following application of a NO donor is decreased, once cardiomyocytes or isolated hearts have been subjected to a preceding period of ischemia/reperfusion [74,75], possibly by a diminished response of guanylyl cyclase to NO.

In conclusion, NO can preserve ischemic blood flow and attenuate platelet aggregation and neutrophil–endothelium interaction following ischemia/reperfusion. Low concentrations of NO also increase cardiomyocyte function. On the contrary, higher NO concentrations depress cardiomyocyte function, mediate inflammatory processes following ischemia/reperfusion, impair mitochondrial respiration and even induce cardiomyocyte death. Thus, NO mediates protective as well as deleterious myocardial effects which are critically dependent on the specific experimental conditions.

	10. Considerations on confounding variables in studies on NO effects

Top Abstract 1. Introduction 2. Production of nitric... 3. Metabolism of nitric... 4. NO-cyclic guanosine... 5. Coronary vascular effects... 6. Inflammation and NO 7. Cardiomyocyte function 8. Cardiomyocyte mitochondria... 9. NO and apoptosis 10. Considerations on... 11. Importance of NO... 12. Importance of NO... 13. Final conclusion References

 
When trying to define the effects of NO on the heart following ischemia/reperfusion, the results are clearly affected by the experimental preparation and setup:

1. Addition of red blood cells to an isolated buffer perfused heart preparation reduces the extent of irreversible tissue injury by delivering NO to the myocardium [79].

2. Addition of neutrophils to an isolated buffer-perfused heart preparation worsens the functional and morphological outcome following ischemia/reperfusion [55], since neutrophil adherence to the vascular endothelium is increased following ischemia/reperfusion [54]. This increased neutrophil adherence is related to loss of NO production during reperfusion. Preservation of endothelial function by administration of L-arginine [56] or a NO donor [57] inhibits neutrophil adherence following ischemia/reperfusion.

3. Administration of heparin to in vivo preparations affects the morphological outcome following ischemia/reperfusion, since heparin abolishes platelet aggregation and subsequently plugging of small vessels [80] and thereby counteracts the adherence and aggregation otherwise seen following a decrease in NO concentration.

4. The ischemia/reperfusion-induced free radical production differs between in vitro buffer-perfused hearts and in situ hearts of anesthetized or awake animals, resulting in a substantially different production of peroxynitrite during reperfusion.

5. Species-dependently different NOS isoforms contribute to a different extent to ischemia/reperfusion injury. In rats, the contribution of NOS1 to ischemia/reperfusion is greater than that of NOS3, while in rabbits the major isoform involved in ischemia/reperfusion injury is NOS3 [11].

6. The time and duration of NOS inhibitor administration (bolus application of a high NOS inhibitor concentration vs. continuous administration of a low NOS inhibitor concentration) lead—even within the same animal species —to opposite effects [81]. This observation is in part explained by a dose-dependent selectivity of NOS inhibitors for the different NOS isoforms.

7. NOS inhibitors such as L-NAME or L-NNA in the presence of vitamin C can release NO at a rate of 1:200 to 1:1000 of the administered inhibitor concentration [82]. Thus, administration of millimolar concentrations of L-NAME or L-NNA may result in the release of micromolar NO concentrations, thereby potentially substituting for the achieved NOS inhibition.

8. Gene knockouts for NOS1/NOS3 in mice to establish their effects in ischemia/reperfusion injury might also be counteracted by compensatory increases in other proteins such as NOS2 [83]. Moreover, using gene chips it was demonstrated that in NOS1 and NOS3 knockout mice 67 and 47 genes, respectively, were expressed differentially compared to the respective wild type mice. Some of the encoded proteins are known to be involved in ischemia/reperfusion injury (e.g., heat shock proteins) [6].

Keeping the above limitations in mind, we now will summarize the existing literature on the importance of NO for the functional and morphological outcome following myocardial ischemia/reperfusion.

	11. Importance of NO for contractile function following myocardial ischemia/reperfusion

Top Abstract 1. Introduction 2. Production of nitric... 3. Metabolism of nitric... 4. NO-cyclic guanosine... 5. Coronary vascular effects... 6. Inflammation and NO 7. Cardiomyocyte function 8. Cardiomyocyte mitochondria... 9. NO and apoptosis 10. Considerations on... 11. Importance of NO... 12. Importance of NO... 13. Final conclusion References

 
In NOS3 knockout mice functional recovery following myocardial ischemia/reperfusion is attenuated (Table 1), while over-expression of NOS3 accelerates functional recovery. Also, pharmacological blockade of NOS attenuates the recovery of left ventricular developed pressure following ischemia/reperfusion in isolated mice and guinea pig hearts. Taken together, these studies in mice and guinea pigs suggest that endogenous NO is cardioprotective and attenuates myocardial stunning.

View this table: [in this window] [in a new window]   Table 1 Effects on functional recovery following myocardial ischemia/reperfusion

 
However, studies in other species using different NOS inhibitors at different concentrations did not unequivocally support the above conclusion. In rats, a dose-dependent effect of NOS inhibitors was observed with low (low micromolar concentrations) or high concentrations (>50 µM) being ineffective or even aggravating myocardial stunning while an intermediate concentration of NOS inhibitors (10–20 µM) improved recovery of contractile function following ischemia/reperfusion. Similar findings were obtained in isolated rabbit hearts, in which low concentrations of NOS inhibitors improved functional recovery while high concentrations of NOS inhibitors had no effect. Finally, in dogs, a low concentration of a NOS inhibitor decreased recovery of regional myocardial function, while higher concentrations of NOS inhibitors either improved or had no effect on functional recovery following ischemia/reperfusion.

While exogenous NO in rabbits and dogs always worsened the functional outcome following ischemia/reperfusion, independent of the approach to increase the NO concentration (inhaled NO, L-arginine, SNAP), in rats the functional recovery following ischemia/reperfusion was either decreased or increased without a clear dose-dependency or relation to the NO donor used.

In conclusion, the importance of NO for the functional recovery following myocardial ischemia/reperfusion appears to be species-dependent. While in guinea pigs and mice endogenous NO mediates beneficial effects, blockade of endogenous NO in rabbits and dogs improves and exogenous NO worsens the functional outcome following myocardial ischemia/reperfusion.

	12. Importance of NO for irreversible tissue injury following myocardial ischemia/reperfusion

Top Abstract 1. Introduction 2. Production of nitric... 3. Metabolism of nitric... 4. NO-cyclic guanosine... 5. Coronary vascular effects... 6. Inflammation and NO 7. Cardiomyocyte function 8. Cardiomyocyte mitochondria... 9. NO and apoptosis 10. Considerations on... 11. Importance of NO... 12. Importance of NO... 13. Final conclusion References

 
NOS3 knockout mice have increased infarct size following myocardial ischemia/reperfusion; however, in some mice strains blockade of the compensatorily over-expressed NOS2 (using specific NOS2 inhibitors) was necessary to demonstrate the detrimental effect of the absence of NOS3 for ischemia/reperfusion injury (Table 2).

View this table: [in this window] [in a new window]   Table 2 Effects on irreversible tissue injury following myocardial ischemia/reperfusion

 
Administration of NO or NO donors shortly before or at the time of reperfusion did not cause adverse effects, but in some instances decreased irreversible tissue injury. Part of the beneficial effect achieved by exogenous NO or NO donors was mediated by attenuated neutrophil adherence to the vascular endothelium [56].

In conclusion, the above data point towards a reduction of irreversible tissue injury by endogenous NO or exogenous NO.

	13. Final conclusion

Top Abstract 1. Introduction 2. Production of nitric... 3. Metabolism of nitric... 4. NO-cyclic guanosine... 5. Coronary vascular effects... 6. Inflammation and NO 7. Cardiomyocyte function 8. Cardiomyocyte mitochondria... 9. NO and apoptosis 10. Considerations on... 11. Importance of NO... 12. Importance of NO... 13. Final conclusion References

 
In summary, when reviewing the existing literature we were surprised by its inconsistency: (1) the lack of characterization of the involved NOS isoforms in myocardial ischemia/reperfusion injury among different animal species; (2) the lack of direct measurements of myocardial NO concentration and/or NOS activity to assure sufficient NOS inhibition (given the different concentrations of NOS inhibitors used); (3) the lack of consideration of non-enzymatic NO production as a potential source of NO; (4) and the absence of plasma or blood components in in vitro studies which impact on the one hand on NO delivery and metabolism and on the other hand on myocardial perfusion, thus making a direct comparison of studies impossible.

Future research in this field will have to focus more precisely on the identification and standardization of potential confounding experimental factors that influence synthesis, transport, and interaction of NO with various targets in tissue and blood.

	Notes

 
Time for primary review 16 days

	References

Top Abstract 1. Introduction 2. Production of nitric... 3. Metabolism of nitric... 4. NO-cyclic guanosine... 5. Coronary vascular effects... 6. Inflammation and NO 7. Cardiomyocyte function 8. Cardiomyocyte mitochondria... 9. NO and apoptosis 10. Considerations on... 11. Importance of NO... 12. Importance of NO... 13. Final conclusion References

 

1. Bolli R. Cardioprotective function of inducible nitric oxide synthase and role of nitric oxide in myocardial ischemia and preconditioning: an overview of a decade of research. J. Mol. Cell Cardiol. (2001) 33:1897–1918.[CrossRef][ISI][Medline]
2. Lefer A.M., Campbell B., Shin Y.-K., et al. Simvastatin preserves the ischemic-reperfused myocardium in normocholesterolemic rat hearts. Circulation (1999) 100:178–184.[Abstract/Free Full Text]
3. Asanuma H., Kitakaze M., Funaya H., et al. Nifedipine limits infarct size via NO-dependent mechanisms in dogs. Basic Res. Cardiol. (2001) 96:497–505.[CrossRef][ISI][Medline]
4. Hartman J.C., Kurc G.M., Hullinger T.G., et al. Inhibition of nitric oxide synthase prevents myocardial protection by ramipril. J. Pharmacol. Exp. Ther. (1994) 3:1071–1076.
5. Hafezi-Moghadam A., Simoncini T., Yang Z., et al. Acute cardiovascular protective effects of corticosteroids are mediated by non-transcriptional activation of endothelial nitric oxide synthase. Nat. Med. (2002) 8:473–479.[CrossRef][ISI][Medline]
6. Schulz R., Cohen M.V., Behrends M., Downey J.M., Heusch G. Signal transduction of ischemic preconditioning. Cardiovasc. Res. (2001) 52:181–198.[Free Full Text]
7. Bolli R. The late phase of preconditioning. Circ. Res. (2000) 87:972–983.[Abstract/Free Full Text]
8. Baxter G.F. Role of adenosine in delayed preconditioning of myocardium. Cardiovasc. Res. (2002) 55:483–494.[Abstract/Free Full Text]
9. Kelm M. Nitric oxide metabolism and breakdown. Biochim. Biophys. Acta (1999) 1411:273–289.[Medline]
10. Becker B.F., Kupatt C., Massoudy P., Zahler S. Reactive oxygen species and nitric oxide in myocardial ischemia and reperfusion. Z. Kardiol. (2000) 89(Suppl. 9):IX88–IX91.[CrossRef]
11. Pabla R., Curtis M.J. Endogenous protection against reperfusion-induced ventricular fibrillation: role of neuronal versus non-neuronal sources of nitric oxide and species dependence in the rat versus rabbit isolated heart. J. Mol. Cell Cardiol. (1996) 28:2097–2110.[CrossRef][ISI][Medline]
12. Brahmajothi M.V., Campbell D.L. Heterogeneous basal expression of nitric oxide synthase and superoxide dismutase isoforms in mammalian heart. Circ. Res. (1999) 85:575–587.[Abstract/Free Full Text]
13. Massion P.B., Balligand J.-L. Modulation of cardiac contraction, relaxation and rate by the endothelial nitric oxide synthase (eNOS): lessons from genetically modified mice. J. Physiol. (2003) 546:63–75.[Abstract/Free Full Text]
14. Barouch L.A., Harrison R.W., Skaf M.W., et al. Nitric oxide regulates the heart by spatial confinement of nitric oxide synthase isoforms. Nature (2002) 416:337–339.[Medline]
15. Sears C.E., Bryant S.M., Ashley E.A., et al. Cardiac neuronal nitric oxide synthase isoform regulates myocardial contraction and calcium handling. Circ. Res. (2003) 92:e52–e59.[Abstract/Free Full Text]
16. Radomski M.W., Palmer R.M.J., Moncada S. Comparative pharmacology of endothelium-derived relaxing factor, nitric oxide and prostacyclin in platelets. Br. J. Pharmacol. (1987) 92:181–187.[ISI][Medline]
17. McCall T.B., Boughton-Smith N.K., Palmer R.M.J., Whittle B.J.R., Moncada S. Synthesis of nitric oxide from L-arginine by neutrophils. Biochem. J. (1989) 261:293–296.[ISI][Medline]
18. De Frutos T., de Miguel L.S., Farré J., et al. Expression of an endothelial-type nitric oxide synthase isoform in human neutrophils: modification by tumor necrosis factor-alpha and during acute myocardial infarction. J. Am. Coll. Cardiol. (2001) 37:800–807.[Abstract/Free Full Text]
19. Sánchez De Miguel L., Arriero M., Farré J., et al. Nitric oxide production by neutrophils obtained from patients during acute coronary syndromes: expression of the nitric oxide synthase isoforms. J. Mol. Cell Cardiol. (2002) 39:818–825.
20. Woditsch I., Schrör K. Prostacyclin rather than endogenous nitric oxide is a tissue protective in myocardial ischemia. Am. J. Physiol. (1992) 263:H1390–H1396.[ISI][Medline]
21. Depre C., Fierain L., Hue L. Activation of nitric oxide synthase by ischaemia in the perfused heart. Cardiovasc. Res. (1997) 33:82–87.[Abstract/Free Full Text]
22. Csonka C., Szilvássy Z., Fülöp F., et al. Classic preconditioning decreases the harmful accumulation of nitric oxide during ischemia and reperfusion in rat hearts. Circulation (1999) 100:2260–2266.[Abstract/Free Full Text]
23. Wang P., Zweier J.L. Measurement of nitric oxide and peroxynitrite generation in the postischemic heart. J. Biol. Chem. (1996) 271:29223–29230.[Abstract/Free Full Text]
24. Giraldez R.R., Panda A., Xia Y., Sanders P., Zweier J.L. Decreased nitric oxide synthase activity causes impaired endothelium-dependent relaxation in the postischemic heart. J. Biol. Chem. (1997) 272:21420–21426.[Abstract/Free Full Text]
25. Amrani M., Chester A.H., Jayakumar J., Schyns C.J., Yacoub M.H. L-Arginine reverses low coronary reflow and enhances postischaemic recovery of cardiac mechanical function. Cardiovasc. Res. (1995) 30:200–204.[Abstract/Free Full Text]
26. Noda K., Sasaguri M., Ideishi M., Ikeda M., Arakawa K. Role of locally formed angiotensin II and bradykinin in the reduction of myocardial infarct size in dogs. Cardiovasc. Res. (1993) 27:334–340.[Abstract/Free Full Text]
27. Mollnau H., Wendt M., Szöcs K., et al. Effects of angiotensin II infusion on the expression and function of NAD(P)H oxidase and components of nitric oxide/cGMP signaling. Circ. Res. (2002) 90:e58–e65.[Abstract/Free Full Text]
28. Gurevitch J., Frolkis I., Yuhas Y., et al. Tumor necrosis factor-alpha is released from the isolated heart undergoing ischemia and reperfusion. J. Am. Coll. Cardiol. (1996) 28:247–252.[Abstract]
29. Frangogiannis N.G., Lindsey M.L., Michael L.H., et al. Resident cardiac mast cells degranulate and release preformed TNF-, initiating the cytokine cascade in experimental canine myocardial ischemia/reperfusion. Circulation (1998) 98:699–710.[Abstract/Free Full Text]
30. Irwin M.W., Mak S., Mann D.L., et al. Tissue expression and immunolocalization of tumor necrosis factor- in postinfarction dysfunctional myocardium. Circulation (1999) 99:1492–1498.[Abstract/Free Full Text]
31. Sanders D.B., Larson D.F., Hunter K., Gorman M., Yang B. Comparison of tumor necrosis factor-alpha effect on the expression of iNOS in macrophage and cardiac myocytes. Perfusion (2001) 16:67–74.[Abstract/Free Full Text]
32. Zweier J.L., Samouilov A., Kuppusamy P. Non-enzymatic nitric oxide synthesis in biological systems. Biochim. Biophys. Acta (1999) 1411:250–262.[Medline]
33. Kitakaze M., Node K., Takashima S., et al. Role of cellular acidosis in production of nitric oxide in canine ischemic myocardium. J. Mol. Cell Cardiol. (2001) 33:1727–1737.[CrossRef][ISI][Medline]
34. Godber B.L.J., Doel J.J., Sapkota G.P., et al. Reduction of nitrite to nitric oxide catalyzed by xanthine oxidoreductase. J. Biol. Chem. (2000) 275:7757–7763.[Abstract/Free Full Text]
35. Feelisch M., Rassaf T., Mnaimneh S., et al. Concomitant S-, N-, and heme-nitros(yl)ation in biological tissues and fluids: implications for the fate of NO in vivo. FASEB J. (2002) 16:1775–1785.[Abstract/Free Full Text]
36. Brovkovych V., Stolarczyk E., Oman J., Tomboulian P., Malinski T. Direct electrochemical measurement of nitric oxide in vascular endothelium. J. Pharm. Biomed. Anal. (1999) 19:135–143.[CrossRef][ISI][Medline]
37. Bolli R., Jeroudi M.O., Patel B.S., et al. Direct evidence that oxygen-derived free radicals contribute to postischemic myocardial dysfunction in the intact dog. Proc. Natl. Acad. Sci. U. S. A. (1989) 86:4695–4699.[Abstract/Free Full Text]
38. Rassaf T., Preik M., Kleinbongard P., et al. Evidence for in vivo transport of bioactive nitric oxide in human plasma. J. Clin. Invest. (2002) 109:1241–1248.[CrossRef][ISI][Medline]
39. Friebe A., Koesling D. Regulation of nitric oxide-sensitive guanylyl cyclase. Circ. Res. (2003) 93:96–105.[Abstract/Free Full Text]
40. Gewaltig M.T., Kojda G. Vasoprotection by nitric oxide: mechanisms and therapeutic potential. Cardiovasc. Res. (2002) 55:250–260.[Abstract/Free Full Text]
41. Kojda G., Kottenberg K. Regulation of basal myocardial function by NO. Cardiovasc. Res. (1999) 41:514–523.[Free Full Text]
42. Agulló L., Garcia-Dorado D., Escalona N., et al. Effect of ischemia on soluble and particulate guanylyl cyclase-mediated cGMP synthesis in cardiomyocytes. Am. J. Physiol. Heart Circ. Physiol. (2003) 284:H2170–H2176.[Abstract/Free Full Text]
43. Du Toit E.F., Meiring J., Opie L.H. Relation of cyclic nucleotide ratios to ischemic and reperfusion injury in nitric oxide-donor treated rat hearts. J. Cardiovasc. Pharmacol. (2001) 38:529–538.[CrossRef][ISI][Medline]
44. Heusch G., Post H., Michel M.C., Kelm M., Schulz R. Endogenous nitric oxide and myocardial adaptation to ischemia. Circ. Res. (2000) 87:146–152.[Abstract/Free Full Text]
45. Bloch W., Mehlhorn U., Krahwinkel A., et al. Ischemia increases detectable endothelial nitric oxide synthase in rat and human myocardium. Nitric Oxide: Biol. Chem. (2001) 5:317–333.[CrossRef][ISI][Medline]
46. Mehlhorn U., Bloch W., Krahwinkel A., et al. Activation of myocardial constitutive nitric oxide synthase during coronary artery surgery. Eur. J. Cardiothorac. Surg. (2000) 17:305–311.[Abstract/Free Full Text]
47. Padilla F., Garcia-Dorado D., Agulló L., et al. Intravenous administration of the natriuretic peptide urodilatin at low doses during coronary reperfusion limits infarct size in anesthetized pigs. Cardiovasc. Res. (2001) 51:592–600.[Abstract/Free Full Text]
48. Ehring T., Baumgart D., Krajcar M., et al. Attenuation of myocardial stunning by the ACE-inhibitor ramiprilat through a signal cascade of bradykinin and prostaglandins, but not nitric oxide. Circulation (1994) 90:1368–1385.[Abstract/Free Full Text]
49. Stamler J.S., Loh E., Roddy M.-A., Currie K.E., Creager M.A. Nitric oxide regulates basal systemic and pulmonary vascular resistance in healthy humans. Circulation (1994) 89:2035–2040.[Abstract/Free Full Text]
50. Huang P.L., Huang Z., Mashimo H., et al. Hypertension in mice lacking the gene for endothelial nitric oxide synthase. Nature (1995) 377:239–242.[CrossRef][Medline]
51. Duncker D.J., Bache R.J. Inhibition of nitric oxide production aggravates myocardial hypoperfusion during exercise in the presence of a coronary artery stenosis. Circ. Res. (1994) 74:629–640.[Abstract/Free Full Text]
52. Ehring T., Krajcar M., Baumgart D., et al. Cholinergic and -adrenergic coronary vasomotion with increasing ischemia–reperfusion injury. Am. J. Physiol. (1995) 268:H886–H894.[ISI][Medline]
53. Kim S.-J., Ghaleh B., Kudej R.K., et al. Delayed enhanced nitric oxide-mediated coronary vasodilation following brief ischemia and prolonged reperfusion in conscious dogs. Circ. Res. (1997) 81:53–59.[Abstract/Free Full Text]
54. Ma X.-L., Weyrich A.S., Lefer D.J., Lefer A.M. Diminished basal nitric oxide release after myocardial ischemia and reperfusion promotes neutrophil adherence to coronary endothelium. Circ. Res. (1993) 72:403–412.[Abstract/Free Full Text]
55. Pabla R., Buda A.J., Flynn D.M., et al. Nitric oxide attenuates neutrophil-mediated myocardial contractile dysfunction after ischemia and reperfusion. Circ. Res. (1996) 78:65–72.[Abstract/Free Full Text]
56. Sato H., Zhao Z.Q., McGee D.S., et al. Supplemental L-arginine during cardioplegic arrest and reperfusion avoids regional postischemic injury. J. Thorac. Cardiovasc. Surg. (1995) 110:302–314.[Abstract/Free Full Text]
57. Lefer D.J., Nakanishi K., Johnston W.E., Vinten-Johansen J. Antineutrophil and myocardial protection actions of a novel nitric oxide donor after acute myocardial ischemia and reperfusion in dogs. Circulation (1993) 88:2337–2350.[Abstract/Free Full Text]
58. Nossuli T.O., Hayward R., Scalia R., Lefer A.M. Peroxynitrite reduces myocardial infarct size and preserves coronary endothelium after ischemia and reperfusion in cats. Circulation (1997) 96:2317–2324.[Abstract/Free Full Text]
59. Wildhirt S.M., Weismueller S., Schulze C., et al. Inducible nitric oxide synthase activation after ischemia/reperfusion contributes to myocardial dysfunction and extent of infarct size in rabbits: evidence for a late phase of nitric oxide-mediated reperfusion injury. Cardiovasc. Res. (1999) 43:698–711.[Abstract/Free Full Text]
60. Woolfson R.G., Patel V.C., Neild Gh., Yellon D.M. Inhibition of nitric oxide synthesis reduces infarct size by an adenosine-dependent mechanism. Circulation (1995) 91:1545–1551.[Abstract/Free Full Text]
61. Minamino T., Kitakaze M., Node K., Funaya H., Hori M. Inhibition of nitric oxide synthesis increases adenosine production via an extracellular pathway through activation of protein kinase C. Circulation (1997) 96:1586–1592.[Abstract/Free Full Text]
62. Obata T. Adenosine production and its interaction with protection of ischemic and reperfusion injury of the myocardium. Life Sci. (2002) 71:2083–2103.[CrossRef][ISI][Medline]
63. Belosjorow S., Bolle I., Duschin A., Heusch G., Schulz R. TNF antibodies are as effective as ischemic preconditioning in reducing infarct size in rabbits. Am. J. Physiol. Heart Circ. Physiol. (2003) 284:H927–H930.[Abstract/Free Full Text]
64. Wagner D.R., McTiernan C., Sanders V.J., Feldman A.M. Adenosine inhibits lipopolysaccharide-induced secretion of tumor necrosis factor- in the failing human heart. Circulation (1998) 97:521–524.[Abstract/Free Full Text]
65. Thielmann M., Dörge H., Martin C., et al. Myocardial dysfunction with coronary microembolization: signal transduction through a sequence of nitric oxide, tumor necrosis factor- and sphingosine. Circ. Res. (2002) 90:807–813.[Abstract/Free Full Text]
66. Vila-Petroff M.G., Younes A., Egan J., Lakatta E.G., Sollott S.J. Activation of distinct cAMP-dependent and cGMP-dependent pathways by nitric oxide in cardiac myocytes. Circ. Res. (1999) 84:1020–1031.[Abstract/Free Full Text]
67. Shah A.M., Spurgeon H.A., Sollott S.J., Talo A., Lakatta E.G. 8-Bromo-cGMP reduces the myofilament response to Ca²⁺ in intact cardiac myocytes. Circ. Res. (1994) 74:970–978.[Abstract/Free Full Text]
68. Naim K.L., Rabindranauth P., Weiss H.R., et al. Positive inotropy due to lowering cyclic GMP is also mediated by increase in cyclic AMP in control and hypertrophic hearts. Can. J. Physiol. Pharmacol. (1998) 76:605–612.[CrossRef]<