title: Levosimendan inhibits interleukin-1β-induced apoptosis through activation of Akt and inhibition of inducible nitric oxide synthase in rat cardiac fibroblasts.
abstract: Levosimendan, a calcium sensitizer, known as an inotropic agent has a cytoprotective effect against apoptosis in cardiomyocytes. However, the cytoprotective effect of levosimendan on cardiac fibroblasts has not been clarified. The aim of this study was to examine whether levosimendan modulates interleukin (IL)-1β-induced apoptosis in adult rat cardiac fibroblast. Cardiac fibroblasts were isolated from adult male Wistar rats. Apoptosis of cardiac fibroblasts was evaluated by 4',6-diamidino-2-phenylindole staining and TdT-mediated d-UTP nick end labeling (TUNEL)-based staining. Expression of inducible nitric oxide (NO) synthase (iNOS) and phosphorylation of Akt (Ser473) were determined by Western blotting. Assessment of NO production in the culture medium was performed by using 4,5-diaminofluorescein as a fluorescent probe. Immunofluorescence staining was performed to examine cytochrome-c translocation. Levosimendan (3-100μM) concentration-dependently inhibited IL-1β (4ng/ml, 24h)-induced apoptotic changes in cardiac fibroblasts, such as cellular shrinkage, nuclear condensation and the increase of TUNEL-positive cells. Levosimendan inhibited IL-1β-induced iNOS expression and subsequent NO production. 1400W, an iNOS inhibitor, suppressed the IL-1β-induced apoptosis. Levosimendan alone-treatment concentration-dependently increased phosphorylation of Akt (Ser473). LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, reversed the protective effect of levosimendan on IL-1β-induced apoptosis in TUNEL assay. In addition, levosimendan and 1400W inhibited the IL-1β-induced cytosolic translocation of cytochrome-c. LY294002 reversed the suppressive effects of levosimendan. The present study for the first time demonstrated that levosimendan inhibits IL-1β-induced apoptosis via the activation of PI3K/Akt pathway and the inhibition of iNOS expression in adult rat cardiac fibroblasts.
authors.affiliation: Laboratory of Veterinary Pharmacology, School of Veterinary Medicine, Kitasato University, Higashi 23 Bancho 35-1, Towada city, Aomori 034-8628, Japan. Electronic address: mokada@vmas.kitasato-u.ac.jp.
authors.forename: Muneyoshi
authors.initials: M
authors.lastname: Okada
authors.affiliation: Laboratory of Veterinary Pharmacology, School of Veterinary Medicine, Kitasato University, Higashi 23 Bancho 35-1, Towada city, Aomori 034-8628, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Yamawaki
mesh_terms: D000578:Amidines / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001596:Benzylamines / Q000494:pharmacology; D002867:Chromones / Q000494:pharmacology; D045304:Cytochromes c / Q000378:metabolism; D019610:Cytoprotection / Q000187:drug effects; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006835:Hydrazones / Q000494:pharmacology*; D053583:Interleukin-1beta / Q000494:pharmacology*; D008297:Male; D009025:Morpholines / Q000494:pharmacology; D009206:Myocardium / Q000166:cytology*; D009569:Nitric Oxide / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D011724:Pyridazines / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects; D000077464:Simendan
chemical_list: D000578:Amidines; D001596:Benzylamines; D002867:Chromones; D004791:Enzyme Inhibitors; D006835:Hydrazones; D053583:Interleukin-1beta; D009025:Morpholines; C104820:N-(3-(aminomethyl)benzyl)acetamidine; D011724:Pyridazines; D009569:Nitric Oxide; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D000077464:Simendan; D045304:Cytochromes c; D052247:Nitric Oxide Synthase Type II; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt
keywords: Akt; Apoptosis; Cardiac fibroblasts; Inducible nitric oxide synthase; Levosimendan