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Cytokine-induced nitric oxide production inhibits mitochondrial energy production and impairs contractile function in rat cardiac myocytes

Tetsuya Tatsumi, MD, PhD^*, Satoaki Matoba, MD^*, Akira Kawahara, MD^*, Natsuya Keira, MD^*, Jun Shiraishi, MD^*, Kazuko Akashi, MD^*, Miyuki Kobara, MD^*, Tetsuya Tanaka, MD^*, Maki Katamura, MD^*, Chiaki Nakagawa, MD^*, Bon Ohta, MD, PhD^*, Takeshi Shirayama, MD, PhD^*, Kazuo Takeda, MD, PhD^*, Jun Asayama, MD, PhD, Henry Fliss, PhD and Masao Nakagawa, MD, PhD^*

^* Second Department of Medicine, Kyoto Prefectural University of Medicine; Kyoto, Japan
Department of Clinical Pharmacology, Kyoto Pharmaceutical University, Kyoto, Japan
Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada

View larger version (19K): [in a new window]   Figure 1 Time course of NO2–/NO3– (NOx) production. Cardiac myocytes were incubated with either (a) 10–4 mol/liter or 10–3 mol/liter sodium nitroprusside (SNP), or (b) 10 ng/ml IL-1ß (IL) in the presence or absence of 3 mmol/liter NG-monomethyl-L-arginine (L-NMMA) for the indicated time, and NOx production in the culture media was measured as described in Methods. Control preparations contained no additives. The interaction between five groups and time course was significant (p < 0.0001). p < 0.0001 vs. control (n = 6).

View larger version (24K): [in a new window]   Figure 2 Time course of lactate production. Cardiac myocytes were incubated with (a) 10–4 mol/liter or 10–3 mol/liter sodium nitroprusside (SNP), (b) 10–4 mol/liter or 10–3 mol/liter 8-bromo cGMP, or (c) 10 ng/ml IL-1ß (IL) in the presence or absence of 3 mmol/liter NG-monomethyl-L-arginine (L-NMMA) for the indicated time, and lactate production was measured in the media as described in Methods. Control preparations contained no additives. The interaction between seven groups and time course was significant (p < 0.0001). p < 0.0001 vs. control (n = 6).

View larger version (54K): [in a new window]   Figure 3 Glucose consumption rate in cardiac myocytes. Myocytes were incubated with (a) 10–4 mol/liter or 10–3 mol/liter sodium nitroprusside (SNP), (b) 10–4 mol/liter or 10–3 mol/liter 8-bromo cGMP, or (c) 10 ng/ml IL-1ß (IL) in the presence or absence of 3 mmol/liter NG-monomethyl-L-arginine (L-NMMA) for 24 h, and glucose consumption rate was measured as described in Methods. Control preparations received no additives. p < 0.0001 vs. control (n = 6).

View larger version (60K): [in a new window]   Figure 4 ATP content in cardiac myocytes. Myocytes were incubated with (a) 10–4 mol/liter or 10–3 mol/liter sodium nitroprusside (SNP), (b) 10–4 mol/liter or 10–3 mol/liter 8-bromo cGMP, or (c) 10 ng/ml interleukin (IL)-1ß in the presence or absence of 3 mmol/liter NG-monomethyl-L-arginine (L-NMMA) for 48 h, and ATP content was measured as described in Methods. Control preparations received no additives. p < 0.01; p < 0.001; p < 0.0001 vs. control (n = 6).

View larger version (49K): [in a new window]   Figure 5 Mitochondrial enzyme activity in cardiac myocytes. Myocytes were incubated in the presence of 10 ng/ml IL-1ß (IL), with or without 3 mmol/liter NG-monomethyl-L-arginine (L-NMMA), and 10–4 mol/liter or 10–3 mol/liter sodium nitroprusside (SNP). The activity of the indicated mitochondrial enzymes was assayed as described in Methods. Control preparations received no additives. All activities are in units of µmol/min/mg protein. p < 0.01; p < 0.0001 vs. control (n = 6).

View larger version (17K): [in a new window]   Figure 6 Electrophysiological effects in cardiac myocytes. Representative traces show membrane potential and calcium current (ICa) in myocytes treated with 10 ng/ml IL-1ß (IL) or 10–3 mol/liter sodium nitroprusside (SNP) as described in Methods.

View larger version (40K): [in a new window]   Figure 7 Peak calcium current in cardiac myocytes. The cells were incubated with 10 ng/ml IL-1ß (IL) in the presence or absence of 10–7 mol/liter KT5823, 5 x 10–4 mol/liter aminoguanidine (AG), and 3 mmol/liter NG-monomethyl-L-arginine (L-NMMA), 10–3 mol/liter 8-bromo cGMP in the presence or absence of 10–7 mol/liter KT5823, or 10–3 mol/liter sodium nitroprusside (SNP) for 48 h, and calcium current (ICa) was measured as described in Methods. p < 0.0001 vs. control; #p < 0.01 vs. 8-bromo-cGMP (n = 13).

View larger version (42K): [in a new window]   Figure 8 Myocyte contractility. The cells were incubated with 10 ng/ml IL-1ß (IL) in the presence or absence of 10–7 mol/liter KT5823, 5 x 10–4 mol/liter aminoguanidine (AG), and 3 mmol/liter NG-monomethyl-L-arginine (L-NMMA), or 10–3 mol/liter sodium nitroprusside (SNP) for 48 h, and cell shortening was measured as described in Methods. p < 0.0001 vs. control (n = 10).