1Department of Clinical Evaluation of Medicines and Therapeutics, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan; and 2Department of Pharmacology, University of South Alabama School of Medicine, Mobile, Alabama 36688
Submitted 22 January 2004 ; accepted in final form 3 June 2004
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ABSTRACT |
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ischemia; cultured cardiomyocytes
The mitochondrial pathway of apoptosis is very tightly regulated. Although most cytoprotective agents prevent cytochrome c release, recent studies have revealed that other steps in the process are subject to regulation. In the present study, we have examined the mechanism underlying the cardioprotective activity of the -amino acid, taurine. Taurine (2-aminoethanesulfonic acid) is a ubiquitous substance involved in osmoregulation, modulation of calcium transport, and regulation of oxidative stress, in particular through its ability to scavenge hypochlorous acid (16, 19). Moreover, taurine has been found to prevent high-glucose-mediated endothelial cell apoptosis through its antioxidant property and regulation of intracellular calcium homeostasis (27). Because osmotic stress, calcium overload, and oxidative stress adversely impact mitochondrial function (25), there is reason to suspect that taurine might benefit the cardiomyocyte through the mitochondrial-linked pathway.
In the present study, the interaction between taurine and mitochondria-mediated apoptosis is investigated in a newly developed simulated ischemia model utilizing isolated cardiomyocytes, which are incubated with medium containing and lacking taurine and then sealed within cultured flasks (21). Our findings show that taurine suppresses simulated ischemia-induced apoptosis in cardiomyocytes by targeting the Apaf-1/caspase-9 apoptosome.
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METHODS |
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Detection of apoptotic cells. To visualize fragmented nuclei, we fixed cells with 1% paraformaldehyde for 30 min at room temperature. After being rinsed in PBS, the cells were permeabilized in 70% ethanol. The cells were rinsed twice in PBS and stained with a fluorescent dye, Hoechst 33258 (Sigma-Aldrich), for 15 min at room temperature. After a final rinse in PBS, the cells were mounted in the FlowFade antifade reagent (Molecular Probes) and visualized under ultraviolet light with the Olympus fluorescence microscopy system. More than 100 cardiomyocytes obtained from 3 different primary culture preparations were counted. The percentage of apoptotic nuclei was calculated as the ratio of fragmented nuclei to the total amount of nuclei. Further evaluation of apoptosis was performed with a commercially available cell death detection kit to find DNA strand breaks using the terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling (TUNEL) reagent according to the manufacturer's protocol (Promega). Cells that showed positive TUNEL staining in the nuclei were identified as apoptotic.
Measurement of mitochondrial membrane potential.
Loss of was assessed using a fluorescent dye, the lipophilic cationic probe JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine; Intergen). Cells were incubated with 5 µg/ml JC-1 for 15 min at 37°C and examined with the Olympus fluorescence microscopy system. The excitation wavelength was 488 nm, and the emission fluorescence for JC-1 was monitored at 530 and 590 nm. The red emission of the dye is attributable to a potential-dependent aggregation in the mitochondria, reflecting
. Green fluorescence reflects the monomeric form of JC-1, appearing in the cytosol after mitochondrial membrane depolarization.
Preparation of mitochondrial and cytosolic fractions and total cell lysates. Mitochondrial and cytosolic fractions were prepared from nontreated or taurine-treated cells cultured under nonischemic or ischemic conditions. Preparation of mitochondrial and cytosolic fractions was achieved using a commercially available mitochondria/cytosol fractionation kit according to the manufacturer's protocol (BioVision). The cytosolic and mitochondrial fractions were stored at 80°C. Total cellular proteins were extracted in lysis buffer containing 150 mM NaCl, 10 mM Tris (pH 7.4), and 1 mM EDTA, plus 1% Triton X-100, 1% deoxycholic acid and protease inhibitor mixture (Sigma-Aldrich), followed by centrifugation at 1,500 g for 20 min. Protein concentrations were determined by the method of Lowry et al. (13), using bovine serum albumin as a standard.
Western blot analysis. Proteins (12 µg) from the mitochondrial fraction, cytosolic fraction, or total cell lysate were analyzed by SDS-PAGE (12.5 or 14% gel). After blotting, the Immobilon-P membrane (Millipore) was blocked with 5% BSA in Tween 20 in PBS at room temperature for 1 h. Immunoblots were incubated at room temperature for 60 min with the specific primary antibody to cytochrome c (PharMingen) or Apaf-1 (BioVision). After further washing, the membranes were incubated for 1 h with the secondary antibody (horseradish peroxidase conjugated). The enhanced chemiluminescence procedure was used for detection of the bands (ECL; Santa Cruz Biotechnology). Blots were reprobed with antibody to actin (Sigma-Aldrich) as a loading control. Quantitative analysis of immunoblotted bands was performed by computer program (NIH Image, version 1.61).
Detection of caspase-3 and -9 immunoreactivity. Total cell lysate (12 µg) was separated by 14% SDS-PAGE and then subjected to Western blot analysis with the use of antibody against caspase-3 or -9 (Santa Cruz Biotechnology).
Immunoprecipitation. For determining the Apaf-1-caspase-9 interaction, the cell lysates were prepared in lysis buffer containing 150 mM NaCl, 10 mM Tris (pH 7.4), and 1 mM EDTA, plus 1% Triton X-100, 1% deoxycholic acid, and protease inhibitor mixture. After homogenization and centrifugation, the supernatants were immunoprecipitated with antibody against Apaf-1 (1:150 dilution; Santa Cruz Biotechnology) plus 10 µl of protein A-Sepharose (Santa Cruz Biotechnology) for 5 h. Immunoprecipitates were washed, separated by 12.5% SDS-PAGE, and then subjected to Western blot analysis with the use of antibody against either Apaf-1 or caspase-9 (Medical & Biological Laboratories).
Determination of ATP content. The ATP content of the myocytes was measured according to the manufacturer's instructions, using a commercially available ATP assay kit (Toyo) The results are expressed in picomoles per microgram of protein. Protein concentration was determined by the method of Lowry et al. (13), using bovine serum albumin as a standard.
Statistical analysis.
Depending on the design of the experiment, statistical significance was determined using the Student's t-test, 2 test, or analysis of variance, with the Bonferroni method being used to compare individual data points for a significant F value. Each value was expressed as a mean ± SE. Differences were considered significant when the calculated P value was <0.05.
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RESULTS |
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DISCUSSION |
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Despite the accumulating data concerning Apaf-1/caspase 9 apoptosome, it remains to be elucidated how taurine inhibits apoptosome formation. There was no difference in the level of intracellular ATP content, cytochrome c release, and Apaf-1 expression between taurine-treated and untreated myocytes after a 24-h ischemic insult. Recent reports (3, 5, 17) have documented that a heat shock protein, HSP70, interacts with Apaf-1 and blocks the assembly of functional apoptosome. To elucidate the involvement of HSP70 in taurine-mediated cytoprotection, the expression of HSP was analyzed by immunoblotting; however, HSP70 was not upregulated by taurine (data not shown). Recently, we have demonstrated that taurine prevents the ischemia-induced apoptosis in cardiomyocytes through Akt (23). Thus it is possible that taurine-mediated activation of Akt negatively regulates Apaf-1/caspase-9 interaction, although further studies are required.
Numerous studies (9, 12, 20, 28) have suggested that Apaf-1 plays a crucial role in mitochondria-mediated apoptosis. Mitochondria-mediated apoptosis is involved in the onset of cardiovascular diseases (4, 14, 15, 26), especially in ischemic heart disease. Taking these findings together with previous reports that the administration of taurine shows efficacy in the treatment of patients with congestive heart failure (1, 2), it could be proposed that analyses of cytoprotective mechanisms of taurine provide novel strategies for the treatment of ischemic heart disease.
In conclusion, our findings show that taurine suppresses ischemia-induced apoptosis in cardiomyocytes by preventing formation of the Apaf-1/caspase-9 apoptosome. This is the first demonstration of the molecular mechanisms for the antiapoptotic effects of taurine.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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