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Published on: April 21, 2010
Nitrotyrosinylation, remodeling and endothelial-myocyte uncoupling in iNOS, cystathionine beta synthase (CBS)
Soumi Kundu1, Munish Kumar, Utpal Sen
1Department of Physiology & Biophysics, University of Louisville School of Medicine, Louisville, Kentucky 40202, USA.
Insights
High homocysteine (Hcy) levels contribute to cardiovascular disease by generating nitrotyrosine, activating matrix metalloproteinases (MMPs), and uncoupling endothelium-myocytes, independent of inducible nitric oxide synthase (iNOS).
Area of Science:
- Cardiovascular Biology
- Nitric Oxide Signaling
- Matrix Remodeling
Background:
- Hyperhomocysteinemia (HHcy) is linked to cardiovascular diseases, but the role of inducible nitric oxide synthase (iNOS) remains controversial.
- Endothelial-myocyte (E-M) coupling is crucial for cardiac function, and its disruption can lead to cardiac dysfunction.
- Nitration of tyrosine residues by reactive nitrogen species is implicated in cardiovascular pathology.
Purpose of the Study:
- To investigate the hypothesis that homocysteine (Hcy) induces nitrotyrosine formation via endothelial nitric oxide synthase, leading to E-M uncoupling.
- To differentiate the roles of iNOS versus constitutive nitric oxide synthases (eNOS, nNOS) in Hcy-mediated nitrotyrosine generation and cardiac matrix remodeling.
- To assess the impact of genetic modifications in cystathionine beta-1 synthase (CBS) and iNOS on cardiac function and structure.
Main Methods:
- Analysis of left ventricular (LV) tissue from wild-type (WT), CBS heterozygote knockout (CBS-/+), iNOS homozygote knockout (iNOS-/-), and double knockout (CBS-/+/iNOS-/-) mice.
- Measurement of nitrotyrosine, matrix metalloproteinases (MMP-2, MMP-9) via zymography, and collagen deposition via trichrome staining.
- Assessment of endothelial-dependent relaxation in cardiac rings to determine E-M function.
Main Results:
- Elevated Hcy in CBS-/+ mice and reduced nitric oxide in iNOS-/- mice were confirmed.
- Nitrotyrosine and MMP-9 levels were significantly increased in CBS-/+ and double knockout mice compared to WT.
- Collagen levels and left ventricular hypertrophy were markedly elevated in double knockouts, indicating a synergistic effect of high Hcy and absence of iNOS, leading to robust E-M uncoupling.
Conclusions:
- Homocysteine induces nitrotyrosine formation near the endothelium, activating MMPs and causing E-M uncoupling, irrespective of iNOS activity.
- The absence of iNOS exacerbates Hcy-induced cardiac dysfunction and matrix remodeling, particularly when combined with elevated Hcy levels.
- These findings clarify the detrimental role of Hcy in cardiovascular pathology and highlight the complex interplay between nitric oxide synthases and matrix remodeling.
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.
