doi: 10.1161/CIRCULATIONAHA.105.588236
(Circulation. 2005;112:3668-3671.)
© 2005 American Heart Association, Inc.
Editorial |
"La Donna è Mobile..."
Is Cardiac Neuronal Nitric Oxide Synthase Such a Disconcerting Enzyme?
From the Unit of Pharmacology and Therapeutics (FATH 5349), Department of Medicine, Université catholique de Louvain, Brussels, Belgium.
Correspondence to J.-L. Balligand, MD, PhD, Unit of Pharmacology and Therapeutics (FATH 5349), Department of Medicine, Université catholique de Louvain, 53 Avenue Mounier, 1200 Brussels, Belgium. E-mail Balligand{at}mint.ucl.ac.be
Key Words: Editorials • contractility • myocardial infarction • nitric oxide synthase • remodeling
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Introduction |
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 Top Introduction Which and Where Are...nNOS and Cardiomyocyte...nNOS in the Ischemic...Where Are We Now?References |
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As a prototypical vasorelaxant, nitric oxide (NO) produced by the endothelium indirectly regulates cardiac function through its modulation of the coronary reserve and peripheral hemodynamics. Since the initial description of a direct, autocrine regulation of cardiomyocyte contraction by a constitutive nitric oxide synthase,1 a substantial body of evidence has confirmed the pleiotropic effects of NO produced within cardiac myocytes on many aspects of their cellular biology, such as oxygen consumption,2 excitation-contraction (EC) coupling,3 and hypertrophic remodeling4 (for a review, see elsewhere5).
Article p 3729
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Which and Where Are the Cardiac Nitric Oxide Synthases? |
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TopIntroductionWhich and Where Are...nNOS and Cardiomyocyte...nNOS in the Ischemic...Where Are We Now?References |
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The endothelial nitric oxide synthase (eNOS; encoded by the NOS3 gene) and the neuronal nitric oxide synthase (nNOS; encoded by NOS1) are the 2 constitutive NOSs expressed in cardiac myocytes. eNOS is enriched in plasmalemmal and T-tubular caveolae,6 where it colocalizes with caveolin-3 (the myocyte-specific structural protein of caveolae). One study7 identified nNOS in sarcoplasmic reticulum (SR) membranes of normal rabbit and human hearts; there is also biochemical evidence for nNOS expression in mitochondria.8 In addition, nNOS is abundantly expressed in cardiac adrenergic and cholinergic nervous fibers, and eNOS is expressed in both endothelial and endocardial cells. On ischemia or sepsis, inflammatory cytokines induce the expression of the calcium-independent NOS (inducible nitric oxide synthase [iNOS], encoded by NOS2) in cardiomyocytes and inflammatory cells infiltrating the myocardium.
At first glance, this multiplicity of NOS isoforms would hardly be compatible with signaling specificity on limited (sub)cellular targets a fortiori given the theoretical diffusibility of NO as a gas. In muscle cells, however, the abundant distribution of cytosolic myoglobin and oxidant radicals continuously produced by muscle contraction is likely to significantly limit NO bioavailability and its ability to diffuse (at least as a free radical) to molecular targets distant from its enzymatic source. This restricted diffusion, combined with the differential subcellular localization of each NOS, confers specificity and efficiency to NO signaling by confining the effects of NO on target proteins colocalized with each isoform. In aortic endothelial and smooth muscle cells, the dynamic formation of such "signalosomes" was supported by the identification of multiprotein complexes assembled by the combined interaction of the chaperone heat shock protein 90 with both eNOS and soluble guanylate cyclase,9 a major downstream effector of NO signaling. It is likely, therefore, that spatial confinement of the constitutive NOS with key proteins regulating EC coupling subserves their modulation of specific aspects of contractility. Accordingly, the use of preferential enzymatic inhibitors and genetic deletion/overexpression of each NOS isoform has helped to decipher their respective influence on contractility, as extensively reviewed elsewhere.5
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nNOS and Cardiomyocyte Contractility |
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TopIntroductionWhich and Where Are...nNOS and Cardiomyocyte...nNOS in the Ischemic...Where Are We Now?References |
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In the normal heart, nNOS is thought to exert a tonic inhibition on EC coupling, as suggested by the increase in basal cardiomyocyte contractility after nNOS preferential inhibition or genetic deletion. This has been attributed to an attenuation of L-type calcium currents by nNOS-derived NO.10 Others, however, have observed unchanged L-type currents in cardiomyocytes from nNOS-deficient mice.11 One difficulty with such a paradigm may be the localization of nNOS in the SR (at least in the normal heart), not on the peripheral or T-tubular sarcolemma, where voltage-dependent calcium channels are (as well as eNOS, which has been proposed by some,12 but not all, to regulate L-type currents). Rather, the SR location of nNOS would make it more suitable for the regulation of calcium uptake by the SR calcium ATPase (SERCA). Indeed, this has been proposed on the basis of an analysis of relaxation properties and diastolic calcium kinetics. nNOS-deficient cardiac myocytes display slower kinetics of cytosolic calcium decay during diastole and altered lusitropic properties.10 NO produced from nNOS in SR membranes may then promote SERCA activity. By the same token, it would also sustain EC coupling by replenishing SR calcium stores (despite tonic inhibition of L-type calcium entry). But this is another point of contention. In 1 study, nNOS-deficient cardiomyocytes had decreased SR calcium stores and were unable to sustain a positive force-frequency response13; in another, SR calcium stores were increased,10 possibly as a result of increased L-type calcium entry.
How then would the inotropic effect of ß-adrenergic (ß-AR) stimulation be affected by nNOS? From the inhibitory effect of nNOS on L-type currents, one would predict a potentiation of the ß-AR response after nNOS inhibition or genetic deletion. In isolated cardiomyocytes, this has been consistently observed at low catecholamine concentrations (<10 nmol/L isoproterenol).14 Again, the results at higher concentrations are divergent in vitro, with a sustained increase in contraction in 1 study15 but decreased contraction in another study.11 More agreement with a consistently decreased inotropic response to ß-AR stimulation may be found in vivo (see the article by Barouch et al11 and the article by Dawson et al16 in this issue). This would suggest that the increase in L-type calcium current may not offset the more profound alteration of calcium cycling (ie, deficient SR calcium loading as a result of decreased SERCA function, as discussed above) in the stressed heart.
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nNOS in the Ischemic Heart |
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TopIntroductionWhich and Where Are...nNOS and Cardiomyocyte...nNOS in the Ischemic...Where Are We Now?References |
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The role of nNOS in the diseased heart has received little attention so far. Some recent observations add another layer of complexity to its regulation of EC coupling in the failing heart. First, in infarcted rats, nNOS protein abundance was shown to be upregulated in both cardiac nerves17 and myocytes.18 In the former, nNOS was shown to restore the sympathovagal balance through a potentiation of muscarinic cholinergic neurotransmission; in the latter cell type, immunohistochemical analysis further showed that nNOS was located mostly at the plasmalemmal membrane, not in the SR. Second, NO produced by nNOS in the SR was shown to downregulate the activity of xanthine oxidoreductase, an important source of oxidant radicals in the failing heart.2 The availability of nNOS–/– mice offered the possibility of examining the functional importance of nNOS regulation in the context of cardiac stress such as myocardial infarction.
In the study reported in the present issue, Dawson et al16 examine the impact of nonconditional nNOS genetic deletion on postinfarction mortality, left ventricular remodeling, and basal and stimulated ß-AR contractility. In their strain, they find no effect of nNOS deletion on mortality when animals are selected for similar infarct sizes (average, 39%). Of interest, a clear sex difference appears with higher mortality in males regardless of the nNOS genotype. This may be at variance with the influence of the eNOS genotype on the sex difference in susceptibility to ischemia.19
Using an original 3D echo measurement, they then found clear signs of adverse remodeling in nNOS–/– hearts with enlarged end-systolic and end-diastolic dimensions at all time points of the study (1, 4, and 8 weeks after infarction). At the end of the study period, the mice underwent invasive hemodynamic measurements of systolic and diastolic function. Consistent with their previous cell contractility studies,10,14 in noninfarcted mice, basal LV function was enhanced in the nNOS–/– group. A slight increase persisted at baseline in infarcted nNOS–/– mice compared with infarcted wild-type (WT) littermates. By inference from their previous cell studies, this is interpreted as the consequence of the removal of the nNOS inhibition on L-type calcium currents, although this was not directly measured here. The inotropic response to ß-AR stimulation is more surprising; in noninfarcted mice, ß-AR stimulation of nNOS–/– mice results in a blunted increase in Ees (ventricular elastance, an index of ventricular performance) compared with WT. This is contrary to previous findings in isolated cardiomyocytes14 and perhaps is unexpected because of the proposed deinhibition of L-type calcium currents after nNOS deletion (see above). The authors propose that this altered inotropic reserve may result from an increased sympathetic tone in nNOS–/– mice, which would also explain the increased basal contractility and the discrepancy with their in vitro cell work in which cells would be withdrawn from such endogenous adrenergic tone. To what extent such an explanation remains valid in the context of profound anesthesia, as used for the catheter measurements, with expected perturbation of the autonomic balance in all groups remains speculative. Another explanation is based on previous observations that nNOS deletion results in higher xanthine oxidase activity2 and oxidant stress likely to decrease cardiac myofilament sensitivity. However, this would be expected to affect the contractility of cardiomyocytes equally in vitro and in vivo, which is not the case. An alternative possibility is that genetic deletion of nNOS would result in a reduced SERCA activity and the inability to sustain an increased inotropic response to catecholamines under full hemodynamic load in vivo despite increased L-type calcium influx, which may sufficiently sustain the inotropic response in unloaded isolated cardiomyocytes. This would be in line with the proposed localization of nNOS in SR membranes and previous demonstrations of decreased SR calcium load in nNOS–/– myocytes13 (see above). Additional measurements of SR loading under stress after nNOS inhibition/deletion are clearly needed to clarify this issue.
In addition to the mechanisms proposed above, a decrease in SERCA activity (and decreased ability to maintain SR calcium load under stress) may also account for the surprising inversion of the inotropic response in nNOS–/– mice after infarction, although a positive, albeit smaller, response is observed in WT mice. Of note, contrary to the present findings in mice, Bendall et al20 found an increase in ß-AR response after preferential nNOS inhibition in infarcted rats in which they initially described an upregulation of cardiomyocyte nNOS. The differential effects of acute inhibition versus chronic, nonconditional genetic deletion (and ensuing adaptive phenotypic changes) may provide some explanation for this discrepancy.
Perhaps another argument in favor of the SERCA hypothesis is the consistent finding of altered diastolic properties in noninfarcted nNOS–/– mice and isolated cells in which the same authors had identified slower kinetics of calcium reuptake during relaxation.10 However, the difference between nNOS–/– and WT disappears in the late postinfarction period in the study reported in the present issue. The authors attribute this leveling off to a lesser development of fibrosis in the nNOS–/– genotype. The underlying mechanisms remain equally undetermined. Although the authors propose that nNOS deletion removes a potential source of oxidant radicals from an uncoupled nNOS (by analogy with eNOS21), this benefit would probably be offset by increased superoxide anions produced by a deinhibited xanthine oxidase as proposed above.
Nevertheless, the finding by Dawson et al16 that nNOS protects against adverse postinfarction remodeling is quite clearly demonstrated and adds to a growing consensus on the protective role of the constitutive NOS in the ischemic myocardium. Similarly, eNOS–/– mice with limited infarcts had worse remodeling, hypertrophy of the remote myocardium, and impaired cardiac function,22 whereas cardiomyocyte-specific eNOS overexpressors,4 but not systemic vascular overexpressors,23 were protected. This again emphasizes the importance of the cellular source of NO and, at least for eNOS, argues for an autocrine signaling preventing adverse remodeling. Whether the protective role of nNOS can similarly be attributed to a direct effect in the myocyte independently of systemic (eg, autonomic) influences needs to be tested in cardiomyocyte-specific nNOS transgenic or conditional knockout animals.
Dawson et al also observed an increase in caveolin-3 proteins in infarcted hearts, as previously shown in failing dog hearts24 and postinfarcted human hearts.25 In the latter study, coimmunoprecipitation of caveolin-3 with nNOS was increased as expected and was taken as an indication of the integration of nNOS within functional signaling modules at the sarcolemma, where it would preferentially inhibit L-type calcium entry. Dawson et al wisely refrain from making such a conclusion in their study. Indeed, because caveolin-3 was shown to be redistributed in cytosolic fractions in the postinfarcted heart,26 coimmunoprecipitation from a whole-cell extract would not necessarily or exclusively reflect interaction at the plasma membrane. A stronger case for nNOS translocation would be built if it were clearly identified in plasmalemmal caveolae by electron microscopy, as was done for eNOS.6 One should also keep in mind that the abundance of caveolin is inversely correlated with NOS activity through the inhibitory allosteric binding of caveolin.27 Therefore, any functional interpretation of these changes should be based on a quantitative assessment of caveolin binding to NOS, together with a measurement of NOS activity in situ whenever possible.
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Where Are We Now? |
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TopIntroductionWhich and Where Are...nNOS and Cardiomyocyte...nNOS in the Ischemic...Where Are We Now?References |
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Clearly, nNOS is not the only cardiac NOS to influence basal contractility and ß-AR response. The specific roles of eNOS and iNOS have been summarized in previous reviews.5,28 Perhaps an important aspect emerging from this study by Dawson et al is that paradigms based on isolated cell measurements may not accurately predict the in vivo phenotype for all NOSs. If one would favor their findings in vivo, then nNOS may be the only isoform that does not attenuate the ß-AR inotropic response but sustains it in both normal and failing hearts. From previous evidence, eNOS and perhaps iNOS would serve as an endogenous "brake" against overstimulation by catecholamines, as confirmed in vivo under cardiomyocyte-specific overexpression of this isoform.6,29 Such opposite effects of the 3 isoforms would provide an explanation for the observation that despite the well-documented upregulation of iNOS and nNOS (and in some cases, eNOS) with human cardiac disease, nonspecific NOS inhibitors have little effect on contractile function in human heart failure.30 This should not deter from efforts to modulate the expression and function of specific isoform(s) to positively influence its/their effects on other aspects of cardiac biology such as protection from oxidant stress, myocardial regeneration, or hypertrophic remodeling, as illustrated here.
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Acknowledgments |
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This work is supported by the Fonds National de la Recherche Scientifique (Belgium), an Action de Recherche Concertée from the Communauté Française de Belgique, a Pole d’Attraction Interuniversitaire from the Politique Scientifique Fédérale, and the Fondation Leducq for a Transatlantic Network. I apologize to the many contributors to the field whose work could not be cited because of space limitations.
Disclosure
None.
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Footnotes |
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The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.
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References |
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TopIntroductionWhich and Where Are...nNOS and Cardiomyocyte...nNOS in the Ischemic...Where Are We Now?References |
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1. Balligand JL, Kelly RA, Marsden PA, Smith TW, Michel T. Control of cardiac muscle cell function by an endogenous nitric oxide signaling system. Proc Natl Acad Sci U S A. 1993; 90: 347–351.
2. Kinugawa S, Huang H, Wang Z, Kaminski PM, Wolin MS, Hintze TH. A defect of neuronal nitric oxide synthase increases xanthine oxidase-derived superoxide anion and attenuates the control of myocardial oxygen consumption by nitric oxide derived from endothelial nitric oxide synthase. Circ Res. 2005; 96: 355–362.
3. Petroff MG, Kim SH, Pepe S, Dessy C, Marban E, Balligand JL, Sollott SJ. Endogenous nitric oxide mechanisms mediate the stretch dependence of Ca2+ release in cardiomyocytes. Nat Cell Biol. 2001; 3: 867–873.[CrossRef][Medline] [Order article via Infotrieve]
4. Janssens S, Pokreisz P, Schoonjans L, Pellens M, Vermeersch P, Tjwa M, Jans P, Scherrer-Crosbie M, Picard MH, Szelid Z, Gillijns H, Van de WF, Collen D, Bloch KD. Cardiomyocyte-specific overexpression of nitric oxide synthase 3 improves left ventricular performance and reduces compensatory hypertrophy after myocardial infarction. Circ Res. 2004; 94: 1256–1262.
5. Massion PB, Feron O, Dessy C, Balligand JL. Nitric oxide and cardiac function: ten years after, and continuing. Circ Res. 2003; 93: 388–398.
6. Massion PB, Dessy C, Desjardins F, Pelat M, Havaux X, Belge C, Moulin P, Guiot Y, Feron O, Janssens S, Balligand JL. Cardiomyocyte-restricted overexpression of endothelial nitric oxide synthase (NOS3) attenuates ß-adrenergic stimulation and reinforces vagal inhibition of cardiac contraction. Circulation. 2004; 110: 2666–2672.
7. Xu KY, Huso DL, Dawson TM, Bredt DS, Becker LC. Nitric oxide synthase in cardiac sarcoplasmic reticulum. Proc Natl Acad Sci U S A. 1999; 96: 657–662.
8. Elfering SL, Sarkela TM, Giulivi C. Biochemistry of mitochondrial nitric-oxide synthase. J Biol Chem. 2002; 277: 38079–38086.
9. Venema RC, Venema VJ, Ju H, Harris MB, Snead C, Jilling T, Dimitropoulou C, Maragoudakis ME, Catravas JD. Novel complexes of guanylate cyclase with heat shock protein 90 and nitric oxide synthase. Am J Physiol Heart Circ Physiol. 2003; 285: H669–H678.
10. Sears CE, Bryant SM, Ashley EA, Lygate CA, Rakovic S, Wallis HL, Neubauer S, Terrar DA, Casadei B. Cardiac neuronal nitric oxide synthase isoform regulates myocardial contraction and calcium handling. Circ Res. 2003; 92: e52–e59.[CrossRef][Medline] [Order article via Infotrieve]
11. Barouch LA, Harrison RW, Skaf MW, Rosas GO, Cappola TP, Kobeissi ZA, Hobai IA, Lemmon CA, Burnett AL, O’Rourke B, Rodriguez ER, Huang PL, Lima JA, Berkowitz DE, Hare JM. Nitric oxide regulates the heart by spatial confinement of nitric oxide synthase isoforms. Nature. 2002; 416: 337–339.[Medline] [Order article via Infotrieve]
12. Han X, Kubota I, Feron O, Opel DJ, Arstall MA, Zhao YY, Huang P, Fishman MC, Michel T, Kelly RA. Muscarinic cholinergic regulation of cardiac myocyte ICa-L is absent in mice with targeted disruption of endothelial nitric oxide synthase. Proc Natl Acad Sci U S A. 1998; 95: 6510–6515.
13. Khan SA, Skaf MW, Harrison RW, Lee K, Minhas KM, Kumar A, Fradley M, Shoukas AA, Berkowitz DE, Hare JM. Nitric oxide regulation of myocardial contractility and calcium cycling: independent impact of neuronal and endothelial nitric oxide synthases. Circ Res. 2003; 92: 1322–1329.
14. Ashley EA, Sears CE, Bryant SM, Watkins HC, Casadei B. Cardiac nitric oxide synthase 1 regulates basal and ß-adrenergic contractility in murine ventricular myocytes. Circulation. 2002; 105: 3011–3016.
15. Sears CE, Ashley EA, Casadei B. Nitric oxide control of cardiac function:is neuronal nitric oxide synthase a key component? Philos Trans R Soc Lond B Biol Sci. 2004; 359: 1021–1044.
16. Dawson D, Lygate CA, Zhang M-H, Hulbert K, Neubauer S, Casadei B. nNOS gene deletion exacerbates pathological left ventricular remodeling and functional deterioration after myocardial infarction. Circulation. 2005; 112: 3729–3737.
17. Takimoto Y, Aoyama T, Tanaka K, Keyamura R, Yui Y, Sasayama S. Augmented expression of neuronal nitric oxide synthase in the atria parasympathetically decreases heart rate during acute myocardial infarction in rats. Circulation. 2002; 105: 490–496.
18. Damy T, Ratajczak P, Robidel E, Bendall JK, Oliviero P, Boczkowski J, Ebrahimian T, Marotte F, Samuel JL, Heymes C. Up-regulation of cardiac nitric oxide synthase 1-derived nitric oxide after myocardial infarction in senescent rats. FASEB J. 2003; 17: 1934–1936.
19. Cross HR, Murphy E, Koch WJ, Steenbergen C. Male and female mice overexpressing the beta(2)-adrenergic receptor exhibit differences in ischemia/reperfusion injury: role of nitric oxide. Cardiovasc Res. 2002; 53: 662–671.
20. Bendall JK, Damy T, Ratajczak P, Loyer X, Monceau V, Marty I, Milliez P, Robidel E, Marotte F, Samuel JL, Heymes C. Role of myocardial neuronal nitric oxide synthase-derived nitric oxide in ß-adrenergic hyporesponsiveness after myocardial infarction-induced heart failure in rat. Circulation. 2004; 110: 2368–2375.
21. Takimoto E, Champion HC, Li M, Ren S, Rodriguez ER, Tavazzi B, Lazzarino G, Paolocci N, Gabrielson KL, Wang Y, Kass DA. Oxidant stress from nitric oxide synthase-3 uncoupling stimulates cardiac pathologic remodeling from chronic pressure load. J Clin Invest. 2005; 115: 1221–1231.[CrossRef][Medline] [Order article via Infotrieve]
22. Scherrer-Crosbie M, Ullrich R, Bloch KD, Nakajima H, Nasseri B, Aretz HT, Lindsey ML, Vancon AC, Huang PL, Lee RT, Zapol WM, Picard MH. Endothelial nitric oxide synthase limits left ventricular remodeling after myocardial infarction in mice. Circulation. 2001; 104: 1286–1291.
23. Jones SP, Greer JJ, van Haperen R, Duncker DJ, de Crom R, Lefer DJ. Endothelial nitric oxide synthase overexpression attenuates congestive heart failure in mice. Proc Natl Acad Sci U S A. 2003; 100: 4891–4896.
24. Hare JM, Lofthouse RA, Juang GJ, Colman L, Ricker KM, Kim B, Senzaki H, Cao S, Tunin RS, Kass DA. Contribution of caveolin protein abundance to augmented nitric oxide signaling in conscious dogs with pacing-induced heart failure. Circ Res. 2000; 86: 1085–1092.
25. Damy T, Ratajczak P, Shah AM, Camors E, Marty I, Hasenfuss G, Marotte F, Samuel JL, Heymes C. Increased neuronal nitric oxide synthase-derived NO production in the failing human heart. Lancet. 2004; 363: 1365–1367.[CrossRef][Medline] [Order article via Infotrieve]
26. Ratajczak P, Damy T, Heymes C, Oliviero P, Marotte F, Robidel E, Sercombe R, Boczkowski J, Rappaport L, Samuel JL. Caveolin-1 and -3 dissociations from caveolae to cytosol in the heart during aging and after myocardial infarction in rat. Cardiovasc Res. 2003; 57: 358–369.
27. Piech A, Massart PE, Dessy C, Feron O, Havaux X, Morel N, Vanoverschelde JL, Donckier J, Balligand JL. Decreased expression of myocardial eNOS and caveolin in dogs with hypertrophic cardiomyopathy. Am J Physiol Heart Circ Physiol. 2002; 282: H219–H231.
28. Schulz R, Rassaf T, Massion PB, Kelm M, Balligand JL. Recent advances in the understanding of the role of nitric oxide in cardiovascular homeostasis. Pharmacol Ther. 2005. E-pub.
29. Champion HC, Georgakopoulos D, Takimoto E, Isoda T, Wang Y, Kass D. Modulation of in vivo cardiac function by myocyte-specific nitric oxide synthase-3. Circ Res. 2004; 94: 657–663.
30. Cotton JM, Kearney MT, MacCarthy PA, Grocott-Mason RM, McClean DR, Heymes C, Richardson PJ, Shah AM. Effects of nitric oxide synthase inhibition on basal function and the force-frequency relationship in the normal and failing human heart in vivo. Circulation. 2001; 104: 2318–2323.
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