| Objectives: To investigate the expression of hypoxic-inducible factor-l (HIF-l) within cardiomyocytes or myocardial tissue following ischemia/scalding and to explore roles of HIF-la expression and its mechanism .Methods: 1. Models: Neonatal murine cardiomyocytes cultured in the mixed gas (1 %O2, 5%CO2, 94%N2) were employed as the hypoxic model. HIF-la interfering fragments were compared and screened. The optimal interference double chains interference RNA (dsRNAi) was constructed and screened effective si- HIF-la-RNAi was transfected .Wistar rats inflicted by 40% TBS A of degree scalding were employed as scalding model. 2. Indices: The contents of ATP, ADP and AMP in the cultured cardiomyocytes were determined with HPLC and the energy load and ATP/ADP ratio were calculated. The change of the content of lactate acid (LA) in the culture media and that of the activity of the key enzymes (HK, PFK, LDH) during glycolysis.The changes of the mRNA content and HIF-la protein withing the cardiomyocytes and myocardial tissue were detected with RT-PCR. and Western blotting The expression and orientation activation of HIF-la protein were observed by immunohistochemistry.Results: 1. The optimal interference fragment sequence of HIF-la was screened as follows:2. The mRNA and protein levels of HIF-l within cardiomyocytes increased following burns and hypoxia.. The HIF-la was immediately expressed in the cytoplasma 30 mins after hypoxia and expressed abundantly in the nucleolus,suggesting a nuclear shift 1 hour after hypoxia. there were obviously decrease of the mRNA and protein levels of HIF-l in si-HIF-l-RNAi group contrast with hypoxia group.3. The activities of glycolysis enzymes HK, PFK and LDH within cardiomyocytes during hypoxia increased significantly. And the content of LA in the culture media increased quickly. Compared with those in the simple hypoxia group,there were no obviously increase of the activities of HK, PFK and LDH in si- HIF-l-RNAi group.the LA content in the culture media increased relatively slow.4. The ATP content in cardiomyocytes decreased during hypoxic settings and remained at relative low level during the following 3-12 hours of hypoxia. The ATP content decreased again to the lowest level during 12 -24 hours of hypoxia. At the same time, the contents of ADP and AMP increased slowly. Compared with those in simple hypoxic group, the ATP content in si- HIF-la-RNAi group decreased quickly to the lowest level during 6-12 hours of hypoxia, while the contents of ADP and AMP increased quickly. The changes of energy load and ATP/ADP ratio exhibited similar to those of ATP.5. The contents of HIF-la mRNA and protein in the left and right ventricular tissues in Wistar rats inflicted with 40% TBS Ascalding increased gradually , and peaked at 12 postburn hours( PBHs).Conclusion:1. It is the first time to construct a model of HIF-la low expression within neonatal murine cardiomyocytes successfully through transfecting ddRNAi expression cassettes.lt is a good model for research the function of HIF-1 all round and deeply.This technique wae more cheap ,easy operation and fast than gene knock-out.2. The increase of the HIF-la protein content expression within cardiomyocytes was induced by hypoxia and was influenced by the time and levels of hypoxia. The HIF-la protein content peaked at half hour post hypoxia within cardiomyocytes. The HIF-l was immediately expressed in the cytoplasma 30 mins after hypoxia and expressed abundantly in the nucleolus,suggesting a nuclear shift 1 hour after hypoxia and it was delayed.3.The activities of intracellular enzymes of glycolysis increased. But the levels ATP and energy load decreased shortly and remained at low level thereafter. Nevertheless, the ADP and AMP contents increased gradually. The energy increased because glycolysis raised to make up the energy deficiency.4.When the expression of HIF-l was inhibited, the increment of the enzymatic activity of glycolysis induced by hypoxia decreased ,the ATP and energy load levels decreased abruptly, which lead...
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