The Mechanism Of Progressive Heart Block In Mice Lacking Urea Transporter B

Objective: Identified UT-B null mice and UT-B wide type mice, To determine the change ofcardiac function in UT-B null mice and study the mechanism.Methods: Genotype identification has been done by genomic DNA PCR; Difference inphysiological feature between UT-B null mice (UT-B -/-) and wild type(UT-B +/+) mice wasstudied: 2 mol/L urea hemolysis test was used to observe the erythrocytic reactiveness in highconcentration urea solution.; The expression UT-B mRNA and protein in heart were detectedby RT-PCR and Western blot.; Surface electrocardiogram (ECG) recording (leadⅡ) wasmeasured in wildtype mice and UT-B null mice at ages of 6 w, 16 w and 52 wrespectively; The expression of Bcl-2 and VDAC mRNA in heart were detected byRT-PCR; Contents of MDA and NO, activity of SOD were detected by Colorimetric methodand Nitrate reductase method.Results: Using UT-B gene knock-out founder mice we raised a great quantity progeny mice.Genotype identification was done by genomic DNA PCR. The results showed that theamplification product of UT-B -/- mice was at 280bp, UT-B+/+ mice was at 400bp.heterozygote mice (UT-B+/-) had two bands at 280 and 400bp. The filial generation ofUT-B+/- mice were three genotypes: UT-B+/+, UT-B+/- and UT-B -/-, and the ratio accordwith Mendel's law of inheritance; When the erythrocyte of UT-B-/- mice add to 2 mol/L ureasolution, the cell shrinked immediately, and then swelled gradually until most erythrocytehemolyzed after 10 min. While the erythrocyte of UT-B+/+ mice add to 2 mol/L urea solution,the cell hemolyzed immediately. There is significant difference between the two genotypemice. This result is accorded with the genotype identification result by genomic PCR. Theresult of RT-PCR displayed that the UT-B mRNA expression in the cadiocyte of UT-B+/+mice, but negative in UT-B-/- mice. The result of Western blot is displayed that there is aabout forty five kilodalton UT-B protein express in cadiocyte of UT-B+/+ mice. ECGrecording showed that the P-R interval was significantly delayed in UT-B null mice (43.5±4.2 ms, 45.5±6.9 ms, 43.8±7.6 ms at ages of 6 w, 16 w and 52 w respectively) vs wildtype mice(38.6±2.9 ms, 38.7±5.6 ms, 38.2±7.3 ms, p＜0.05). Atrial ventricular heart block typeⅡandⅢonly appeared in the aging UT-B null mice (52 w old). There are no difference in the heartexpression of Bcl-2 and VDAC mRNA between UT-B -/- mice and UT-B +/+ mice. Theexpression level of VDAC mRNA in heart tissue in UT-B -/- mice is lower than UT-B +/+mice, Contents of MDA and NO, activity of SOD were detected by Colorimetric method andNitrate reductase method. Contents of NO and activity of T-SOD in the aging UT-B-/-micewere lower than that in aging UT-B+/+ mice, But the content of MDA in aging UT-B-/-micewas higher than that that in aging UT-B+/+ mice. The reduction of contents of NO in agingUT-B-/-mice perhaps is the results of down regulation of NOS activity by accumulation ofurea in heart and blood. The reduction of NO and T-SOD in the aging UT-B-/-mice inducelipidic peroxidatic reaction and increase contents of MDA, and the intracellular over load ofCa2+. It will get myocardium damaged in the aging UT-B-/-mice, and make the membranebioelectricity to unstabily. Then affect the excitability and conductivity of cadiocyte.Conclusion:On the basis of building the UT-B genotype identification system (DNA-PCR and 2mol/L urea solution hemolysis test) successfully; UT-B mRNA and protein expressed incadiocyte of UT-B+/+ mice. But it negative expressed in the cadiocyte of UT-B-/-mice; ECGfeature revealed that progressive heart block happened in UT-B -/-mice; The expression ofVDAC mRNA is degraded, It maybe one of the possible reason of progressive heart blockhappened in UT-B -/-mice. Contents of NO,MDA and activity T-SOD is changed in UT-B-/-mice. It maybe another possible reason of progressive heart block happened in UT-B-/-mice.