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Serofendic Acid, a Novel Substance Extracted From Fetal Calf Serum, Protects Against Oxidative Stress in Neonatal Rat Cardiac Myocytes

Toshihiro Takeda, MD^_*, Masaharu Akao, MD, PhD^_*,_*, Madoka Matsumoto-Ida, MD^_*, Masashi Kato, MD^_*, Hiroyuki Takenaka, MD^_*, Yasuki Kihara, MD, PhD^_*, Toshiaki Kume, PhD, Akinori Akaike, PhD and Toru Kita, MD, PhD^_*

^_* Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, Kyoto, Japan
Department of Pharmaceutical Science, Kyoto University Graduate School of Pharmacology, Kyoto, Japan.

View larger version (38K): [in a new window]   Figure 1 (A) The terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining (left panels) and the nuclear counterstaining by 4',6-diamidino-2-phenylindole (DAPI) (right panels) in neonatal rat cardiac myocytes. C = control cells; H = cells exposed to 100 µmol/l H2O2 for 16 h; SFA = cells pretreated with 100 µmol/l serofendic acid for 30 min followed by 100 µmol/l H2O2 for 16 h. Scale bars = 20 µm. (B) Quantitative determination of TUNEL-positive nuclei (n = approximately 3 to 4 for each group from two independent cultures). An average of 200 to 400 nuclei from random fields was analyzed in each sample. (C) Cellular viability evaluated by MTS assay (n = 13 for each group from two independent cultures). The H2O2 treatment was 3 h in this experiment. *p < 0.05 versus group H.

View larger version (30K): [in a new window]   Figure 2 Mitochondrial inner membrane potential (m) in neonatal rat cardiac myocytes. (A) FL-2 histograms of fluorescence-activated cell sorter data from tetramethylrhodamine ethyl ester (TMRE)-loaded cells are shown. H = cells exposed to 100 µmol/l H2O2 for 1 h; SFA = cells pretreated with 100 µmol/l SFA for 30 min followed by 100 µmol/l H2O2 for 1 h. In all of the histograms, the position of the major population of control group is indicated by a vertical dashed line. Results are representative data from at least three independent experiments. (B) Representative data of the percentage of cells that maintain high (>300) TMRE fluorescence. Cells were pretreated with various concentrations of SFA for 30 min, followed by 100 µmol/l H2O2 for 1 h. Serofendic acid preserved m in a concentration-dependent manner. (C) Summarized data of the percentage of cells that maintain high (>300) TMRE fluorescence. Cells were pretreated with various drugs for 30 min followed by 100 µmol/l H2O2 for 1 h. SFA = 100 µmol/l SFA; DZ = 100 µmol/l diazoxide; 5-HD = 500 µmol/l 5-hydroxydecanoate. *p < 0.05 versus H. #p < 0.05 versus corresponding 5-HD–absent group. Other abbreviations as in Figure 1.

View larger version (66K): [in a new window]   Figure 3 Time-lapse analysis of m loss in neonatal rat cardiac myocytes. (A) Representative sequential images of TMRE fluorescence in each group. H = cells exposed to 50 µmol/l H2O2 for 1 h; SFA = cells pretreated with 100 µmol/l serofendic acid for 30 min followed by 50 µmol/l H2O2 for 1 h. Scale bars at 0 min frames: 20 µm. (B) Time course of TMRE fluorescence of 25 cells randomly selected in each group. Similar results were obtained in three independent experiments. (C) Mean fluorescence intensity from 25 cells randomly and prospectively selected in each group. *p < 0.05 versus H at the end of the experimental period. Other abbreviations as in Figure 1. Please see the for an accompanying video to this figure.

View larger version (66K): [in a new window]   Figure 4 Time-lapse analysis of intracellular reactive oxygen species production in neonatal rat cardiac myocytes. (A) Representative sequential images of chloromethyl-2,7-dichlorodihydrofluorescein diacetate (DCF) fluorescence in each group. H = cells exposed to 50 µmol/l H2O2 for 30 min; SFA = cells pretreated with 100 µmol/l serofendic acid for 30 min followed by 50 µmol/l H2O2 for 30 min. Scale bars at 0 min frames: 20 µm. (B) Time course of DCF fluorescence of 25 cells randomly selected in each group. Similar results were obtained in three independent experiments. (C) Mean fluorescence intensity from 25 cells randomly and prospectively selected in each group. *p < 0.05 versus H at the end of the experimental period. Other abbreviations as in Figure 1. Please see the for an accompanying video to this figure.

View larger version (66K): [in a new window]   Figure 5 Time-lapse analysis of intracellular mitochondrial matrix calcium in neonatal rat cardiac myocytes. (A) Representative sequential images of rhod-2 (Molecular Probes, Eugene, Oregon) fluorescence in each group. H = cells exposed to 50 µmol/l H2O2 for 1 h; SFA = cells pretreated with 100 µmol/l serofendic acid for 30 min followed by 50 µmol/l H2O2 for 1 h. Scale bars at 0 min frames: 20 µm. (B) Time course of rhod-2 fluorescence of 25 cells randomly selected in each group. Similar results were obtained in three independent experiments. (C) Mean fluorescence intensity from 25 cells randomly and prospectively selected in each group. *p < 0.05 versus H at the end of the experimental period. Other abbreviations as in Figure 1. Please see the for an accompanying video to this figure.