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. 2009 Jan 1;106(1):119-26.
doi: 10.1002/jcb.21982.

Nitrotyrosinylation, remodeling and endothelial-myocyte uncoupling in iNOS, cystathionine beta synthase (CBS) knockouts and iNOS/CBS double knockout mice

Soumi Kundu  1 , Munish Kumar, Utpal Sen, Paras K Mishra, Neetu Tyagi, Naira Metreveli, David Lominadze, Walter Rodriguez, Suresh C Tyagi
Affiliations

Nitrotyrosinylation, remodeling and endothelial-myocyte uncoupling in iNOS, cystathionine beta synthase (CBS) knockouts and iNOS/CBS double knockout mice

Soumi Kundu et al. J Cell Biochem. .

Abstract

Increased levels of homocysteine (Hcy), recognized as hyperhomocysteinemia (HHcy), were associated with cardiovascular diseases. There was controversy regarding the detrimental versus cardio protective role of inducible nitric oxide synthase (iNOS) in ischemic heart disease. The aim of this study was to test the hypothesis that the Hcy generated nitrotyrosine by inducing the endothelial nitric oxide synthase, causing endothelial-myocyte (E-M) coupling. To differentiate the role of iNOS versus constitutive nitric oxide synthase (eNOS and nNOS) in Hcy-mediated nitrotyrosine generation and matrix remodeling in cardiac dysfunction, left ventricular (LV) tissue was analyzed from cystathionine beta synthase (CBS) heterozygote knockout, iNOS homozygote knockout, CBS-/+/iNOS-/- double knockout, and wild-type (WT) mice. The levels of nitrotyrosine, MMP-2 and -9 (zymographic analysis), and fibrosis (by trichrome stain) were measured. The endothelial-myocyte function was determined in cardiac rings. In CBS-/+ mice, homocysteine was elevated and in iNOS-/- mice, nitric oxide was significantly reduced. The nitrotyrosine and matrix metalloproteinase-9 (MMP-9) levels were elevated in double knockout and CBS-/+ as compared to WT mice. Although MMP-2 levels were similar in CBS-/+, iNOS-/-, and CBS-/+/iNOS-/-, the levels were three- to fourfold higher than WT. The levels of collagen were similar in CBS-/+ and iNOS-/-, but they were threefold higher than WT. Interesting, the levels of collagen increased sixfold in double knockouts, compared to WT, suggesting synergism between high Hcy and lack of iNOS. Left ventricular hypertrophy was exaggerated in the iNOS-/- and double knockout, and mildly increased in the CBS-/+, compared to WT mice. The endothelial-dependent relaxation was attenuated to the same extent in the CBS-/+ and iNOS-/-, compared to WT, but it was robustly blunted in double knockouts. The results concluded that homocysteine generated nitrotyrosine in the vicinity of endothelium, caused MMP activation and endothelium-myocyte uncoupling. The generation of nitrotyrosine was independent of iNOS.

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Figures

Fig. 1
Fig. 1
Genotype of iNOS (A), and CBS (B): Tail tissue DNA was analyzed for iNOS and CBS using respective primers by PCR. In iNOS, wild-type (+/+) copy at kb and disrupted DNA copy (−/−) at 6.3 kb was observed. In CBS, 1.5 kb DNA was observed in wild-type, however, in heterozygote (−/+) two DNA bands were observed.
Fig. 2
Fig. 2
Phenotype of iNOS−/− by the measurements of NO2/NO3 (B) and CBS−/+ by the measurements of Hcy (A) in tail vein blood. The plasma was analyzed by Griess method for NO and Hcy by HPLC and spectrophotometer. N = 6 in each group; *P <0.05 compared with iNOS−/− for NO2/NO3 levels, and compared with CBS−/+ for Hcy levels. **P <0.05 compared with wild-type.
Fig. 3
Fig. 3
The LV tissue homogenates was analyzed for total 3-nitrotyrosine levels. A: Representative the band at 66 kDa in SDS–PAGE followed by Western blot analysis using 3-nitrotyrosine antibody was shown as marker of nitrityrosinylation. Identical amount of total protein was loaded onto each lane. Membranes were re-probed for beta actin. B: Scanned bands unit data of total 3-nitrityrosine/actin ratio in double KO (iNOS−/−/CBS−/+), CBS−/+, iNOS−/−, and wild-type (iNOS+/+/CBS+/+) mice. N = 6 in each group; *P <0.05 compared with iNOS−/− or with CBS−/+ group. **P <0.05 compared with wild-type.
Fig. 4
Fig. 4
The LV tissue homogenates was analyzed for MMP activity by gelatin gel zymography. A: The bands at 92 and 72 kDa for MMP-9 and -2, in SDS–PAGE-zymography, were analyzed for MMP activity. Identical amount of total protein was loaded onto each lane. Identical gels were blotted for beta actin. B: Scan unit data of MMP-2 and -9 activity normalized with actin in double KO (iNOS−/−/CBS−/+), CBS−/+, iNOS−/−, and wild type (iNOS+/+/CBS+/+) mice. N = 6 in each group; *P <0.05 compared with iNOS−/− or with CBS−/+ group. **P <0.05 compared with wild type. ***P <0.05 compared with WT.
Fig. 5
Fig. 5
Histological analysis of the hearts from (1) double KO (iNOS−/−/CBS−/+), (2) CBS−/+, (3) iNOS−/−, and (4) wild-type (iNOS+/+/CBS+/+) mice. The 10 μm paraffin imbedded tissue sections were stained by Trichrome-blue for collagen.
Fig. 6
Fig. 6
A: Biochemical analysis to total hydroxyl praline collagen, and (B) LVH by myocyte size measured by a micro meter in: (1) double KO (iNOS−/−/CBS−/+), (2) CBS−/+, (3) iNOS−/−, and (4) wild-type (iNOS+/+/CBS+/+) mice. N = 6 in each group; *P <0.05 compared with iNOS−/− or with CBS−/+ group. **P <0.05 compared with wild-type. ***P <0.05 compared with WT.
Fig. 7
Fig. 7
A typical experiment of E-M function in cardiac rings: The LV rings were stretched, rested, and activated by calcium. The bradykinin (BK) was used to determine the endothelial-dependent (EDHF) cardiotonic agent to relax the cardiac rings from double KO (iNOS−/−/CBS−/+) and WT (iNOS+/+/CBS+/+) mice. Note: the ring from double KO has no response (tone) to BK, where as ring from WT relaxed normally with BK.
Fig. 8
Fig. 8
The dose response curves: (A) for bradykinin (BK); (B) for acytylcholine (ACH); and (C) for sodium nitroprusside (Npr) in cardiac rings from (1) double KO (iNOS−/−/CBS−), (2) CBS −, (3) iNOS−/−, and (4) wild-type (iNOS+/+/CBS+/+) mice. N = 6 in each group; *P <0.05 compared with iNOS−/− or with CBS−/+ group.
Fig. 9
Fig. 9
Hypothesis suggesting a plausible mechanism of generation of nitrotyrosine in the vicinity of endothelium and the formation of fibrosis in between E and M, causing E-M uncoupling.
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