| The heart failure is a kind of serious heart disease, the mortality rate isextremely high. The happening of the heart failure is indicated thatcardiovascular disease develops to the certain serious degree, which causes toweaken the cardiac muscle shrinkage force or handicaps the diastole function,reduces the heart row of blood quantity。 cannot meet the need of the organicorganization cell metabolism, at the same time the venous blood backflow isblocked, the ecchymosis of the vein system appears, blood dynamics and thenerve-body fluid are caused to be changed. These processes not only cause thecircumference resistance and the ventricle load to be increased, the heart rateto speed up with the increase of the cardiac muscle energy consumption, butalso can cause the cardiac muscle to be large, the lack of the blood, theabnormally rapid heart rate, the decrease of the cardiac muscle cell, the largepromotion of the cardiac muscle and the reorganization of the structure and soon. These factors further cause to worsen the heart failure. The heart failurecreates the changes of the heart interior and the environment causes the changeof the cardiac muscle cell electricity physiological nature, the arrhythmia and asudden death. Therefore, the further research on the change of the cardiacmuscle cell electricity physiological nature at the time of the heart failure, willdeepen our understanding of the heart sudden death mechanism, then help toprovide the better method of preventing the heart sudden death of the patientwith the heart failure.As the excitability cell, the cardiac muscle cell has its own characteristiclong action potential duration. It's the reaction to the repolarization process ofthe cardiac muscle. The massive empirical data demonstrated, the reasonscausing the heart failure are the pressure, the volume load, heredity,metabolism the lack of blood in the cardiac muscle, the blockage of the heartstem, chronic tachycardia and so on. In the model of the animal with the heartfailure, the ventricle cell and the tissue of the heart have the prolongation ofthe characteristic action potential duration. Similarly, the prolongation ofaction potential duration also exists in the ventricle organization and the cell ofthe patient with the heart failure. This indicated regardless of in the cell levelor in the organic level, the change of the electricity physiology the heartfailure is the prolongation of the action potential duration(action potentialduration, APD) which can cause the unstable repolarization process, thencause the disorder of the cardiac muscle electricity physiological process.The prolongation of APD without the changes of any other electric currentdynamics and the electricity attribute archery target is the characteristicelectrophysiology change in the heart failure. Some report demonstrated, indifferent spots of the mammal heart action potential duration is different. Inthe heart failure because of the different prolongation of APD in the differentspots of the mammal cardiac muscle, the speed of the cardiac musclerepolarization is not even, and causes the reentry arrhythmias.The electrophysiology research proved that in the heart failure the decline ofthe ventricle muscle cell K+ electric current is the main reason causing theprolongation of APD. Several evidences demonstrated that transient outwardpotassium current, Ito can affect the action potential duration. Through theanimal model and sample analysis in human cardiac muscle may draw such aconclusion: the formation of the cardiac muscle cell Ito is mainly due topotassium ion channel Kv4.3. In the heart failure Ito declines, at the same time,potassium ion channel Kv4.3 representation drops.In the research on the mechanism in the heart failure, one viewpoint is thatpotassium ion channel Kv1.4 also has the vital role in the formation of thecardiac muscle cell Ito. In order to analyze the function of the potassium ionchannel in the heart failure, we made a reseach on the representation changesof potassium ion channel protein Kv4.3, Kv1.4 and the Mink subsuit in thecardic muscle the dog the animal model with the heart failure. The resultsshowed, in the heart failure, Kv4.3 obviously drops, assists Mink subsuit alsohasthe slight drop, but Kv1.4 rises. That Explained that Kv4.3 plays the mainrole in the change of Ito, at the same time Kv1.4 and Kv4.3 and the Minksubsuit also change differently. So it can be inferred that in the heart failure,the decline of Ito is a complex electrophysiology change process. Besides themain function of Kv4.3, many ions channel protein the representation changeinvolve in it. Therefore the research on the regulation rule of the relatedpotassium ion channel gene representation regarding the declines of Ito will behelpful in exposing the molecular foundation on which f Ito is formed.Through the diaphragm pliers technology the analysis of the single cell ionchannel indicated that in the heart failure the cardiac muscle cell Ito obviouslydeclined, the single ion channel potassium electric current hardly changed, butthe ion channel quantity reduced actually, That may prove the Ito decline iscaused by the reduction of the ion channel representation, not by the change ofthe potassium ion channel electricity physiology attribute. In the human'schronic heart failure and the animal's heart failure model, Ito drops at the sametime Kv4.3 gene mRNA content and the protein representation also drops.These findings all explained that in the heart failure hKv4.3 generepresentation dropsIn order to analyze the representation regulation of the hKv4.3 gene, wehave cloned the hKv4.3 gene 5 ' the non-code area sequence, through directedthe thing to extend the law to determine the transcriptional start site of hKv4.3in the final translation start site was located upstream ATG 4614bp place,moreover it was only one spot. Through the reporter gene method, we studythe hKv4.3 gene function sequence by deleting the sudden change in the5'non-code area sequence. The research discovered the smallest promoterfunction area of hKv4.3 starts in this section of sequence from thetranscriptional start site upstream 156bp the to the transcriptional start site. Inthe smallest promoter function area, we also discovered several adjustmentparts including CArG-box and CACC-box, and CArG-box is important in theactivity of the promoter, but only CACC-box is insufficient to complete thecopy process. In the smallest promoter function area, there is the suppressionelement T (-27 ~ -17bp), its sequence is AGGGGTAAAC. When the Telement was deleted, ,the activeness of promoter doubled. But that has notdiscovered in the the suppression elements in other genes with similarsequence. E-box is located in the smallest promoter upstream but it did notseem to affect the promoter activeness.In order to analyze the expression of hKv4.3 gene, we have cloned andsequenced the 5'untranslated region of the hKv4.3 gene. We identified thetranscriptional start site (TSS) located approximately 4614bp upstream of thefirst exon with primer extension method and the TSS is only one. We study thefunction sequence of hKv4.3 gene 5'-noncode sequence through deletionmutation method with reporter plasmid. A minimal promoter is presentbetween -158bp 5' to the transcriptional start site. Several regulatory elementsin the minimal promoter were identified including a CArG box that wasessential for promoter activity, CACC box that by itself is not sufficient fortranscription, and a novel repressor element designated T (-27 to -17) with thesequence TGGGGTAAAC. Delete T element, the promoter activity increase todouble and no same suppression element in other gene. An E-box wasidentified 5' upstream from the minimal promoter but does not affect theactivity of the minimal promoter. The minimal promoter is TATA-less, acharacteristic feature shared by voltage gated potassium channels including therat KCNC1, the mouse KCNA4,the mouse KCNJ2 and KCNA5 andcharacteristic of constitutively expressed "housekeeping gene". Unidentifiedrepressors exist from -1646 to -834 but are not clearly identified. No cell-typespecific elements were present in the minimal promoter or surroundingelements.Outside of the promoter region a segment from +2 to 160 functionsas an intragenic suppressor of promoter activity and is designated S160.The regulation of hKv4.3 is likely to involve the combinatorial action of,among others, upstream repressor elements, promoter elements, and intragenicsilencers. E-boxes are important cis-acting elements in the regulation ofexpression of genes in striated muscle. In skeletal muscle, an E-box in the type1 sodium channel (SkM1), when bound by myogenic basic helix-loop-helixproteins attenuates the action of upstream repressor elements thus conferringtissue specificity. However, based on our data the E-box in hKv4.3 has no rolein increasing promoter activity or conferring specificity of transcription indifferent cell lines. This is similar to KCNJ2 where an E-box is present withinthe minimal promoter but mutations within the E-box confer no statisticallysignificant changes in the level of expression [9]. CArG boxes are anotherelement that have been identified in a number of structural genes in heart andskeletal muscle (REFS). This motif is required for the induction of severalpromoters in a-adrenergically mediated cardiac growth but does not mediatecell-type specific regulation of expression. Two unique elements in the hKv4.3promoter, T and S160 suppress transcription in a cell-independent manner.Negative elements have been identified in other voltage gated potassiumchannels. The Kv1.5 gene contains a dinucleotide repeat element(poly(GT)19(GA) 1(CA)15(GA)16) which is responsible for silencer activity,binding a 27-kDA KRE (Kv1.5 repressor element) binding factor. A 5'untranslated region from +350 to +158 coupled to a weak enhancr and from-123 to -71 conferred cell-type expression in the Kv3.1 gene. While we havenot identified any cell-type specific elements, it may be likely that furtherelements of the 5' untranslated region of hKv4.3 will play a significant role intissue-specific expression. S element isn't the basic reason for the tissue-typeexpression of hKv4.3 gene.Through the research on the expression regulation of hKv4.3 gene, weidentified the TSS, found the promoter region and element that it is importantto gene expression in hKv4.3 gene. We study the function of these sequences.This is the newest result about the hKv4.3 expression regulation in gene level.It is in favor of explaining the molecular mechanism of heart sudden death anddesigning new medicine according to Ito correlation protein for heart failure . |
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