Homocysteine-dependent cardiac remodeling and endothelial-myocyte coupling in a 2 kidney, 1 clip Goldblatt hypertension mouse model Neetu Tyagi, Karni S. Moshal, David Lominadze, Alexander V. Ovechkin, and Suresh C. Tyagi Abstract: Accumulation of interstitial collagen (fibrosis) between the endothelium and myocytes is one of the hall- marks of cardiac failure in renovascular hypertension (RVH). Renal insufficiency increases plasma homocysteine (Hcy), and levels of peroxisome proliferator-activated receptor-γ (PPAR-γ) are inversely related to plasma Hcy levels. We hy- pothesize that in RVH, accumulation of collagen between the endothelium and myocytes leads to endothelial-myocyte disconnection and uncoupling, in part, by hyperhomocysteinemia. Furthermore, we hypothesize that Hcy increases reac- tive oxygen species, generates nitrotyrosine, activates latent matrix metalloproteinase, and decreases the levels of endo- thelial nitric oxide in response to antagonizing PPAR-γ. To create RVH in mice, the left renal artery was clipped with 0.4-mm sliver wire for the 2 kidney, 1 clip (2K1C) method. Sham surgery was used as a control. To induce PPAR-γ,8 μg/mL ciglitazone (CZ) was administered to drinking water 2 days before surgery and continued for 4 weeks. Mice were grouped as 2K1C, sham, 2K1C+CZ, or sham+CZ (n = 6 in each group). Plasma Hcy increased 2-fold in the 2K1C-treated group (p < 0.05) as compared with the sham, and CZ had no effect on Hcy levels as compared to the 2K1C-treated group. Hcy binding in cardiac tissue homogenates decreased in the 2K1C-treated group but was substan- tially higher in the CZ-treated group. Cardiac reactive oxygen species levels were increased and endothelial nitric oxide were decreased in the 2K1C-treated group. Matrix metalloproteinase-2 and -9 activities were increased in the 2K1C- treated group compared with the control. Levels of cardiac inhibitor of metallopoteinase were decreased, whereas there was no change in tissue inhibitor of metalloproteinase-1 expression in the 2K1C-treated group vs. the sham-treated group. Collagen and nitrotyrosine levels were increased in the 2K1C-treated group, but mice treated with CZ showed lower levels comparatively. Cardiac transferase deoxyuridine nick-end labeling-positive cells were increased, and muscle cells were impaired in the 2K1C-treated mice vs. the sham-control mice. This was associated with decreased acetylcho- line and bradykinin responses, which suggests endothelial-myocyte uncoupling in 2K1C-treated mice. Our results sug- gest that fibrosis between the endothelium and myocytes leads to an endothelial-myocyte disconnection and uncoupling by Hcy accumulation secondary to increased reactive oxygen species, nitrotyrosine, matrix metalloproteinase, and de- creased endothelial nitric oxide in response to antagonizing PPAR-γ. Key words: ECM, collagen, elastin, cystathione β synthase, nitric oxide, arteriosclerosis, renal mechanism. 594 Résumé : L’accumulation de collagène interstitiel (fibrose) entre l’endothélium et les myocytes est un signe d’insuffisance cardiaque dans l’hypertension rénovasculaire (HRV). L’insuffisance rénale augmente l’homocystéine (Hcy) plasmatique, et les taux du récepteur-γ activé par les proliférateurs des peroxysomes (PPAR-γ) sont inversement liés aux taux de Hcy plasmatique. Nous supposons que l’accumulation de collagène entre l’endothélium et les myocy- tes mène à la séparation et au découplage endothélium-myocyte, en partie, par hyperhomocystéinémie. De plus, nous posons que l’Hcy augmente les espèces oxygénées radicalaires, produit de la nitrotyrosine, active la métalloprotéinase matricielle latente et diminue les taux de monoxyde d’azote endothélial en réponse à l’antagonisme du PPAR-γ. Pour induire l’HRV, nous avons posé un clip (fil d’argent de 0, 4 mm) sur l’artère rénale gauche de souris (2R1C). Nous avons utilizé une chirurgie fictive comme témoin. Pour induire le PPAR-γ, nous avons administré du ciglitazone (CZ), à raison de 8 μg/mL, dans l’eau de boisson deux jours avant la chirurgie pour une période de 4 semaines. Nous avons réparti les souris comme suit 2R1C, fictive, 2R1C+CZ, fictive + CZ (n=6 dans chaque groupe). L’Hcy plasmatique a augmenté d’un facteur 2 chez le groupe 2R1C (p < 0,05) comparativement à la valeur témoin, et le CZ n’a pas eu d’effet sur les taux de Hcy. La fixation de Hcy dans les homogénats des tissus cardiaques a diminué chez le groupe 2R1C mais a augmenté considérablement après le traitement au CZ. Les taux des espèces oxygénées radicalaires car- diaques ont augmenté et ceux de monoxyde d’azote endothélial ont diminué chez le groupe 2R1C. Les activités des Can. J. Physiol. Pharmacol. 83: 583–594 (2005) doi: 10.1139/Y05-047 © 2005 NRC Canada 583 Received 23 November 2004. Published on the NRC Research Press Web site at http://cjpp.nrc.ca on 20 July 2005. N. Tyagi, K.S. Moshal, D. Lominadze, A.V. Ovechkin, and S.C. Tyagi. 1,2 Department of Physiology and Biophysics, University of Louisville School of Medicine, Louisville, KY 40202, United States. 1 Corresponding author (e-mail: s0tyag01@louisville.edu). 2 Present address: University of Louisville School of Medicine, Department of Physiology & Biophysics, 500 South Preston Street, Louisville, KY 40202. United States.