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Cardiovascular Research 2002 55(2):225-228; doi:10.1016/S0008-6363(02)00465-0
© 2002 by European Society of Cardiology
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Copyright © 2002, European Society of Cardiology

Response to: endomyocardial nitric oxide synthase and the hemodynamic phenotypes of human dilated cardiomyopathy and of athlete's heart

Jamie T Stark, David J Schaeffer and David R Gross*

Department of Veterinary Biosciences, College of Veterinary Medicine, University of Illinois, Urbana–Champaign, 3516 VMBS Bldg., 2001 S. Lincoln Ave., Urbana, IL 61802, USA

* Corresponding author. Tel.: +1-217-333-2506; fax: +1-217-244-1652 dgross{at}cvm.uiuc.edu

Received 7 May 2002; accepted 7 May 2002

See article by Bronzwaer et al. [15] (pages 270–278) in this issue.

The nitric oxide synthase (NOS) family of enzymes consists of three protein products from three distinct genes that catalyze the conversion of L-citrulline to L-arginine in the presence of NADPH and O2 yielding the diffusible free radical gas NO. The current viewpoint is that two of these genes NOS1 (nNOS) and NOS3 (eNOS) are constitutively expressed while NOS2 (iNOS) is readily inducible. In addition, the neuronal (nNOS) and endothelial (eNOS) proteins contain flexible calmodulin binding domains making these low-output enzymes sensitive to Ca2+ activation. Conversely, the inducible (iNOS) protein binds calmodulin tightly, making this enzyme Ca2+ insensitive. However, this protein is readily induced by numerous cytokines (IL-1β, TNF{alpha}, IFN{gamma}, IL-6) as well as lipopolysaccharide (LPS) [1]. NOS1 and NOS3, by nature of their calcium sensitive, transient activity, mediate cell signaling events associated with NO. NOS2 is a high output enzyme (100–1000-fold greater activity than NOS1 and/or NOS3), immune inducible, calcium insensitive and is typically associated with the response to infection and inflammation [2].

In the normal mammalian heart, all three NOS gene products are expressed. Alterations in expression occur in response to a number of pathological stimuli. NOS1 is located in nerve terminals and may be involved in the synaptic transmission of norepinephrine during normal function [3]. Due to limited available data, the role for NOS1 in pathological settings has yet to be defined. NOS2 is located in endothelial cells, endocardial cells, and cardiomyocytes and seems to play a major role in normal immune function. Modulation of NOS2 expression has been implicated in numerous forms of cardiac pathophysiology, the general consensus being that high levels of NOS2 are associated with dysfunction. NOS3 is expressed in endocardium, coronary endothelium, and cardiomyocytes. It is primarily involved in local blood flow regulation. NOS3 has also been implicated in various forms of cardiac pathophysiology, however, while some have reported increased levels [4,5], others have observed a decrease [6], or no change [7]. Due to its inducible nature, and high flux characteristics, NOS2 has received the majority of attention as being a contributing factor in cardiac dysfunction.

In the last decade, more than 400 articles have been published addressing the role of NOS2 in the heart. Of these, more than 100 have dealt directly with the role of NOS2 in heart failure in both human and animal models. Based on the available data, what seems clear is that NOS2 is increased at all levels, mRNA, protein, and activity, during heart failure [5,7–10]. Sustained increases in NOS2 appear to correlate with increased dysfunction and mortality in the failing myocardium. However, constitutive cardiac-specific over-expression of NOS2 in transgenic mice did not, by itself, confer severe cardiac dysfunction. The explanation for this was the observation of drastic alterations in the substrate, L-arginine, and metabolite, L-citrulline, pools [11]. Feng et al. [9] demonstrated that transgenic mice lacking NOS2 had significantly increased function and survival compared to wild-type animals 30 days post-myocardial infarction. Sam et al. [12] showed that NOS2–/– mice had increased contractile function, decreased apoptosis, and decreased mortality 4 months following myocardial infarction. However, animals studied at 1 month by these investigators did not display these attributes compared to infarcted wild-type controls. Taken together, these data suggest that the timing of NOS2 expression may play a vital role in heart failure models.

Although limited, the data from human clinical subjects provides additional insight into the potential roles of NOS in heart disease. Many studies have shown an increase in NOS2 in pathologic myocardium [5,8,10,13,14] including the article by Bronzwaer et al. in this issue of Cardiovascular Research [15]. These authors were able to correlate NOS2 mRNA levels to physiologically relevant cardiac functional parameters. Previously the same laboratory was able to demonstrate that NOS2 mRNA expression correlated with left ventricular stroke volume (LVSV), left ventricular stroke work (LVSW), and ejection fraction (EF) in a cohort of non-ischemic dilated cardiomyopathy patients [13]. They also demonstrated that NOS3 gene expression correlated to LVSV and LVSW. In the study reported in this issue they have gone one step farther by including a subset of three highly trained cyclists [15]. The focus of the current report is an attempt to demonstrate a relationship between functional left ventricular compliance and levels of NOS2 and NOS3. When NOS2 expression was normalized to left ventricular end-diastolic wall stress (LVEDWS) and plotted against a stiffness modulus, athletes were concentrated at a high NOS2/LVEDWS ratio with a low stiffness modulus while data points from heart failure patients were distributed linearly towards an increased stiffness modulus for a given NOS2/LVEDWS ratio. These data suggest that the remodeling occurring in the athlete's heart represents the positive end of a continuum extending to the dilation and failure associated with increased wall stiffness.

The previous study by the Bronzwaer group was able to demonstrate an increase in the significance of their correlations when exclusion criteria were set [13]. When only patients with moderate LV dysfunction were included, as evidenced by left ventricular end-diastolic pressures (LVEDP) >16 mmHg, an increase in significance of positive correlations between measurements of NOS2 mRNA and EF were shown. However, Kalra et al. [10] demonstrated that decreases in nitrate levels were correlated to increases in ejection fraction in patients who had undergone, and were recovering from bypass surgery. We found this discrepancy interesting when viewed in the context of the transgenic animal studies, and in the context that EF is the most promising surrogate end point for clinical heart failure studies [16]. Given that temporal expression and activity of NOS probably plays a significant role in the development and progression of heart failure, we decided to reanalyze the correlation between NOS2 and NOS3 mRNA levels and the stiffness modulus by normalizing the NOS levels to the EF.

When NOS2 and NOS3 mRNA levels were normalized to EF and plotted against the stiffness modulus from the Bronzwaer [15] data, only the NOS3 gene showed a significant correlation (r = 0.62, P = 0.0001) (Fig. 1). NOS2/EF plotted against the stiffness modulus showed no significant correlation (r = 0.22, P = 0.2166) (Fig. 2). The significant correlation suggests that an increase in NOS3 relative to EF is linearly related to increased wall stiffness. To further explore this notion, we reassessed the information in previous studies of NOS expression in human heart failure, noting the severity of dysfunction in the patient population, and paying particular attention to functional changes that occurred over time and the location from which the biopsies were taken. Haywood et al. [14] measured NOS2 mRNA and protein levels in patients of varying degrees of heart failures (NYHA II–IV). When patients were pooled according to NYHA class, NOS2 expression was more prevalent in class II heart failure than class III (P<0.05) or IV (P<0.02) and was independent of etiology. Multiple biopsies were taken from all four chambers in this study. Although not significant, the authors suggested a trend for higher NOS2 expression in the LV. NOS3 levels were not assessed in this study. Wildhirt et al. [8] assessed the level of NOS2 mRNA and protein in septal biopsies from transplant patients and compared expression levels to coronary flow velocity reserve (CFVR). Notably, in patients who had decreased CFVR at 1 month post-op, but recovered by 12 months post-op, NOS2 mRNA levels decreased during this time, as did transcardiac NO levels and immunoreactivity to nitrotyrosine protein. Conversely, in patients who did not recover function, markers of NOS2 remained elevated compared to those that did recover CFVR. Again, NOS3 levels were not assessed in this study. Recently, Kalra et al. [10] measured mRNA levels of NOS2 and TNF{alpha} in LV biopsies from patients with hibernating myocardium scheduled for bypass surgery. Their patient cohort had an average NYHA score of 2.7 and demonstrated increases in expression of NOS2 and TNF{alpha} mRNA in biopsies taken from hibernating myocardium compared to biopsies taken from normal myocardium. The levels of expression from hibernating regions of the LV were intermediate between normal and ischemic tissue. The decrease in serum nitrite levels was significantly correlated to the change in EF (r = –0.92, P<0.001) again suggesting that a reduction in NO is a prerequisite for recovery of function. Unfortunately, NOS3 was not assayed in this study.


Figure 1
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  		Fig. 1 Linear regression analysis of the relationship between NOS3, normalized by EF, and the stiffness modulus as defined by Bronzwaer et al. Triangles indicate patients with EF>30%, circles patients with EF 20–30%, and squares patients with EF<20%. Correlation coefficient for the line is 0.6226, the P = 0.0001.

 

Figure 2
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  		Fig. 2 Linear regression analysis of the relationship between NOS2, normalized by EF, and the stiffness modulus. Triangles indicate patients with EF>30%, circles patients with EF 20–30% and squares patients with EF<20%. Correlation coefficient for the line is 0.2210, the P = 0.2166.

 
Although many studies that assessed moderate levels of LV dysfunction did not include the measurement of NOS3, studies that assessed severe LV dysfunction have documented alterations in NOS3 expression. Fukuchi et al. [5] measured NOS2 and NOS3 protein expression and activity from both the RV and LV in patients with class III–IV heart failure. Although regional variances existed, increased NOS3 protein expression was observed in the subendocardial areas of the LV. No significant correlations to EF or other hemodynamic parameters were reported, however, analysis was performed based on a scoring system rather than raw data, and trends in the data suggest that re-analysis based on normalization by EF may yield different conclusions. Although increased NOS2 expression was present, it did not correlate with any hemodynamic variables, rather there was a significant correlation between NOS2 expression and the number of infiltrating macrophages (r = 0.992, P<0.01). This reaffirms the idea that NOS2 is more tightly coupled to the immune response than to functional parameters. In failing human hearts from patients with class IV heart failure, Stein et al. [4] measured NOS2 and NOS3 mRNA and protein and found an increase in NOS3 mRNA and protein in cardiomyocytes in all hearts. NOS2 mRNA was only detected in minimal amounts from two of 30 hearts studied, and NOS2 protein was not detected in any. NOS3 mRNA was increased in cardiomyocytes ~200% in all forms of heart disease, and NOS3 protein levels were expressed in cardiomyocytes approximately twofold higher in all hearts, suggesting a tight coupling of transcription/translation for this gene product.

In the study of Bronzwaer et al. [15] the increased expression of NOS2 in the athlete's heart suggests that NOS2 may play a role in beneficial remodeling of the myocardium. Given that these athletes were described as ‘highly trained’ the presence of NOS2 in the myocardium, and its ability to correlate to beneficial LV functional parameters, indicates that this gene may be more involved in the compensatory responses to LV dysfunction than to the pathological progression to end stage failure. Moldoveanu et al. [17], indicate that the cytokine response to exercise training induces increases in plasma concentrations of IL-1, IL-6, and TNF{alpha} in almost any exercise paradigm used. This suggests that the cytokine sensitive NOS2 may be involved in beneficial remodeling of the myocardium. Indeed, reassessing the data available on human tissue by NYHA functional class supports this argument, along with animal studies that measured compensation over longer time courses. The marked prevalence of NOS3 in class IV (end stage) myocardium challenges the idea that the NOS3 gene is constitutively expressed, and suggests that additional studies need to be performed to adequately dissect the role of these two gene products in the evolution of chronic heart failure. Forstermann et al. [6] indicate that the NOS3 promoter region contains 18 putative transcription factor binding sites lending itself to potential regulation by a number of cellular stress factors including: IL-6, NF-{kappa}B, PEA3, INF{gamma}, shear stress, and acute phase proteins.

The story of NOS and heart failure is far from complete. We find the attempt to correlate NOS mRNA to LV function by Bronzwaer et al. [15] very thought provoking, and therefore sought to put this idea into the context of a more clinical indicator of the severity of ventricular dysfunction. The re-analysis of their data, coupled with a reassessment of the data available from other studies investigating human pathological myocardium, led to our alternative interpretations of this study. It is not our intention to challenge the ideas put forth by these authors. We wish to provoke reassessment of current concepts about the roles of NOS2 and NOS3 in human heart failure.


   References
Top
 References
 

  1. Kelly R.A., Balligand J.L., Smith T.W. Nitric oxide and cardiac function. Circ Res (1996) 79:363–380.[Free Full Text]
  2. Nathan C., Qiao-wen X. Nitric oxide synthases: roles, tolls, and controls. Cell (1994) 78:915–918.[CrossRef][Web of Science][Medline]
  3. Schwartz P., Diem R., Dun J.N., Forstermann U. Endogenous and exogenous nitric oxide inhibits norepinephrine release from rat heart sympathetic nerves. Circ Res (1995) 77:841–848.[Abstract/Free Full Text]
  4. Stein B., Eschenhagen T., Rudiger J., et al. Increased expression of constitutive nitric oxide synthase III, but not inducible nitric oxide synthase II, in human heart failure. J Am Coll Cardiol (1998) 32:1179–1186.[Abstract/Free Full Text]
  5. Fukuchi M., Hussain S., Giaid A. Heterogeneous expression and activity of endothelial and inducible nitric oxide synthases in end-stage human heart failure. Circulation (1998) 98:132–139.[Abstract/Free Full Text]
  6. Forstermann U., Boissel J., Kleinert H. Expressional control of the ‘constitutive’ isoforms of nitric oxide synthase (NOS I and NOS III). FASEB J (1998) 12:773–790.[Abstract/Free Full Text]
  7. Takimoto Y., Aoyama T., Keyamura R., et al. Differential expression of three types of nitric oxide synthase in both infarcted and non-infarcted left ventricles after myocardial infarction in the rat. Int J Cardiol (2000) 76:135–145.[CrossRef][Web of Science][Medline]
  8. Wildhirt S.M., Weis M., Schulze C., Conrad N., et al. Expression of endomyocardial nitric oxide synthase and coronary endothelial function in human cardiac allografts. Circulation (2001) 104(suppl. I):I336–I343.[Web of Science][Medline]
  9. Feng Q., Lu X., Jones D.L., et al. Increased inducible nitric oxide synthase expression contributes to myocardial dysfunction and higher mortality after myocardial infarction in mice. Circulation (2001) 104:700–704.[Abstract/Free Full Text]
  10. Kalra D.K., Zhu X., Ramchandani M.K., et al. Increased myocardial gene expression of tumor necrosis factor-alpha and nitric oxide synthase-2. Circulation (2002) 105:1537–1540.[Abstract/Free Full Text]
  11. Heger J., Godeche Z., Flogel U., et al. Cardiac-specific overexpression of inducible nitric oxide synthase does not result in severe cardiac dysfunction. Circ Res (2002) 90:93–99.[Abstract/Free Full Text]
  12. Sam F., Sawyer D.B., Xie Z., et al. Mice lacking inducible nitric oxide synthase have improved left ventricular contractile function and reduced apoptotic death late after myocardial infarction. Circ Res (2001) 89:351–356.[Abstract/Free Full Text]
  13. Heymes C., Vanderheyden M., Bronzwaer J. Endomyocardial nitric oxide synthase and left ventricular preload reserve in dilated cardiomyopathy. Circulation (1999) 99:3009–3016.[Abstract/Free Full Text]
  14. Haywood G.A., Tsao P.S., von der Leyen H.E., et al. Expression of inducible nitric oxide synthase in human heart failure. Circulation (1996) 93:1087–1094.[Abstract/Free Full Text]
  15. Bronzwaer J., Zeitz C., Visser C.A., Paulus W.J. Endomyocardial nitric oxide synthase and the hemodynamic phenotypes of human dilated cardiomyopathy and of athlete's heart. Cardiovasc Res (2002) 55:270–278.[Abstract/Free Full Text]
  16. Anand I.S., Florea V.G., Fisher L. Surrogate end points in heart failure. J Am Coll Cardiol (2002) 39:1414–1421.[Abstract/Free Full Text]
  17. Moldoveanu A.I., Shephard R.J., Shek P.N. The cytokine response to physical activity and training. Sports Med (2001) 31:115–144.[CrossRef][Web of Science][Medline]

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