| Endothelin (ET), a family of vasoconstrictive peptides, distributes in the cardiovascular system, the actions of which are mediated by ETA and ETB receptors, both present in the myocardium and coronary arteries. Myocardial hypoxia/ischemia is a potent stimulus for synthesis of ET-1, a primary isoform of the ET family. Increased endogenous ET-1 tends to aggravate myocardial ischemia via coronary constriction. It was reported that exogenous ET-1 was arrhythmogenic, and ETA receptor antagonist was able to reduce infarct size, improve function of the ischemic myocardium, and decrease arrhythmia incidences during reperfusion, though these findings have not been reported in acute ischemia. Whether ET-1, especially endogenous ET-1, is arrthymogenic or antiarrthymic during acute myocardial ischemia remains controversial. 10-23 deoxyribozyme is a short single strain DNA composed of a catalytic core, flanked by 2 substrate-recognition domains, and cleaving particular RNA substrates. To clarify the role of ET-1 in arrhythmia incidences during acute ischemia, 10-23 deoxyribozymes cleaving ET-1 mRNA were selected in vitro and after being transfected into cultured rat neonatal cardiomyocytes. Ischemic arrhythmias elicited by occlusion of the left anterior descending branch of coronary artery(LAD) in isolated perfused rat hearts and the effects of 10-23 deoxyribozymes were analyzed during 60-min occlusion of LAD. Statistical analysis was performed using ANOVA and Mann-Whitney U, depending on the type of data. P values below 0.05 were considered significantMethods and Results1. Design and selection of ET-1 10-23 deoxyribozymes in vitro(1) ET-1 10-23 deoxyribozymes was composed of a catalytic core of 15deoxynucleotides, flanked by 2 substrate-recognition domains of 9 deoxynucleotideswith two phosphorothioate groups at 5' and 3' end. The cleaving sites were all A-Uand G-U junctions between the initial codon and +219 nucleotide. Five 10-23deoxyribozymes whose free energy was less than -24kcal/mol as DZ1-DZ5 were chosen.(2) Rat ET-1 fall sequence cDNA was amplificated by RT-PCR. 32P-labeled ET-1 RNA substrate was transcripted in vitro, and purified by denaturing polyacrylamide gel electrophoresis(PAGE), followed by electroelution and ethanol precipitation. The purified substrate dentified by autoradiogaphy was homogenized to prevent the affect of inhomogeneity of the transcripted RNA substrate.(3)RNA substrate (5umol/L) and 10-23 deoxyribozymes(lumol/L) was incubated for 1 h at 37, separated by denaturing PAGE and then analyzed by autoradiogaphy. It was shown that DZ2, DZ3, DZ4 and DZ5 cleaved RNA substrate in 1 h, and among them DZ4 was the most efficient one, whose 1-h cleavage was 83.5%. It was also shown that the free energy difference of the two recognition domains of DZ4 was the largest and its cleavage was also the highest, and the difference of the two recognition domains of DZ3 was the smallest and its cleavage was also the lowest.2. Effects of DZ4 on ET-1 expression in cultured neonatal rat cardiomyocytes(1) FAM-labeled DZ4 was applied to detect its uptake by cultured neonatal rat cardiomyocytes. No fluorescence was observed in cardiomyocytes after directly adding FAM-labeled 0.2umol/L DZ4 into the medium, while 12 hours after liposome transfection, fluorescence appeared and was stronger 24 hours after transfection. Intracellular fluorescence transfected by calcium-phosphate was stronger than by liposome 1 2 and 24 hours after transfection.(2) Srum was used to induce hypertrophy in cultured neonatal rat cardiomyocytes. The content of ET-1 mRNA was detected by RT-PCR after administration of DZ4. Twenty-four hours after liposome transfection, semi-quantitative RT-PCR showed that the ratio of ET-1 band density to that of p-actin was 0.615?.044 in the untransfected group. The ratio of 0.lumol/L DZ4 group was 0.566?.046, and there was no significant difference compared to that of the untransfected group and that of the corresponding liposome group(0.617?.050, n=5 . p>0.05) while the ratio of...
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