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OBJECTIVE: To explore the effects of microtubule depolymerization (MD) on the spontaneous beating rate, action potential (AP), and oxygen consumption of cardiac myocytes in rats and its mechanism. METHODS: One-hundred and eighty neonatal SD rats divided into 12 batches were used in the experiment, and 15 rats in each batch were sacrificed for the isolation and culture of cardiac myocytes after the heart tissues were harvested. The cardiac myocytes were respectively inoculated in one 12-well plate filled with 6 round cover slips, one 12-well plate filled with 6 square cover slips, two cell culture flasks, and two cell culture dishes. After routine culture for three days, the cardiac myocytes from all the containers were divided into normal control group (NC, routinely cultured with 3 mL DMEM/F12 solution rewarmed at 37 °C for 3 h) and group MD (routinely cultured with 3 mL DMEM/F12 solution rewarmed at 37 ° and containing 8 µmol/L colchicine for 3 h) according to the random number table, with 3 holes, 1 flask, or 1 dish in each group. The morphological changes in microtubules were observed with confocal laser scanning microscope after immunofluorescent staining. The content of polymerized or dissociative α-tubulin was determined by Western blotting. Spontaneous beating rate of the cells was observed and calculated under inverted microscope. Dissolved oxygen concentration of DMEM/F12 solution containing cardiac myocytes was determined by oxygen microelectrode system before and after the addition of colchicine. Additionally, dissolved oxygen concentration of DMEM/F12 solution and colchicine + DMEM/F12 solution was determined. The whole-cell patch-clamp technique was used to record AP, delayed rectifier K+ current (I(K)), and L-type Ca2+ current (I(Ca-L)) in cardiac myocytes; current density-voltage (I-V) curves were drawn based on the traces. Data were processed with independent or paired samples t-test. RESULTS: (1) In group NC, microtubules of cardiac myocytes were around the nucleus in radial distribution with intact and clear linear tubiform structure. The microtubules in group MD were observed in dispersive distribution with damaged structure and rough linear tubiform structure. (2) In group MD, the content of dissociative α-tubulin of cells (0.61 ± 0.03) was obviously higher than that in group NC (0.46 ± 0.03, t = -6.99, P < 0.05), while the content of polymerized α-tubulin (0.57 ± 0.04) was significantly lower than that in group NC (0.88 ± 0.04, t = 9.09, P < 0.05). (3) Spontaneous beating rate of cells was (59 ± 8) times per min in group MD, which was distinctly higher than that in group NC [(41 ± 7) times per min, t = 5.62, P < 0.01]. (4) Dissolved oxygen concentration of DMEM/F12 solution containing cardiac myocytes was (138.4 ± 2.5) µmol/L, and it was reduced to (121.7 ± 3.6) µmol/L after the addition of colchicine ( t = 26.31, P < 0.05). There was no obvious difference in dissolved oxygen concentration between DMEM/F12 solution and colchicine + DMEM/F12 solution (t = 0.72, P > 0.05). (5) Compared with that of group NC, AP morphology of cells in group MD changed significantly, with unobvious repolarization plateau phase and shorter action potential duration (APD). The APD20, APD50, and APD90 were respectively (36.2 ± 3.8), (73.7 ± 5.7), and (115.1 ± 8.0) ms in group MD, which were significantly shorter than those of group NC [(40.2 ± 2.3), (121.4 ± 7.0), and (169.4 ± 5.6) ms, with t values respectively 2.61, 15.88, and 16.75, P values below 0.05]. (6) Compared with that of group NC, the I-V curve of I(K) of cells in group MD moved up with higher current density under each test voltage (0 to 40 mV) after activation ( with t values from 2. 70 to 3. 76, P values below 0.05) . (7) There was not much alteration in current density of I(Ca-L) under each test voltage (-30 to 50 mV) between 2 groups (with t values from -1.57 to 1.66, P values above 0.05), and their I-V curves were nearly overlapped. CONCLUSIONS: After MD, the I(K) is enhanced without obvious change in I(Ca-L), making AP repolarization faster and APD shortened. Then the rapid spontaneous beating rate increases oxygen consumption of cardiac myocytes of rats.
Subject(s)
Microtubules/metabolism , Mitochondria, Heart/metabolism , Myocytes, Cardiac/metabolism , Action Potentials , Animals , Cells, Cultured , Energy Metabolism , Rats , Rats, Sprague-Dawley , Tubulin/metabolismABSTRACT
Objective To investigate the roles of mitochondrial DNA (mtDNA) in the respiratory function of cerebral mitochondria in rats exposed to acute hypoxia by observing the changes of mitochondrial respiratory function and cytochrome C oxidase (COX) activity. Methods The rat cerebral cortex mitochondria were isolated by centrifugation. Mitochondrial respiratory function and COX activity were measured by Clark oxygen electrode. Results ① Compared with the control group (C), hypoxia group (H) showed significantly elevated state 4 respiration (ST4), decreased state 3 respiration (ST3), and respiratory control rate (RCR). ② ST3 in group of treatment with chloramphenicol (CAP) plus hypoxia (MH) was significantly lower than that in Group C, while ST4 in Group MH was significantly lower than that in groups C and H. RCR in Group MH was lower than that in Group C, but higher than that in Group H. ③ COX activity in Group H was significantly lower than that in Group C. In Group MH, COX activity was higher than that in Group H, but was still lower than that in Group C. Conclusion The complete expression of mtDNA may play an important role in mitochondrial respiratory function. CAP treatment might be beneficial to the recovery of rat respiratory function.
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Objective To investigate the changes and roles of hypoxia inducible factor 1?(HIF 1?) expression in myocardial tissues in rats following severe scald. Methods Male Wistar rats inflicted with 40% TBSA Ⅲ degree scald were used as animal models. HIF 1? protein in myocardial tissues was detected by Western blot and immunohistochemical technique. Results HIF 1? protein expression in rat myocardial tissues increased significantly in the early stage following scald. The difference in HIF 1? level between the left and right ventricles was significant. The increased HIF 1? protein was mainly located in the nucleus. Conclusion HIF 1? protein expression in myocardial tissues of rats can be induced by severe scald and HIF 1? protein expression in the left ventricle is significantly higher than that in the right ventricle. The increased HIF 1? protein in the nucleus can induce downstream cytokine expression.
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Objective To construct recombinant adenovirus vector containing Rattus norvegicus microtubule-associated protein 4 gene,and transfect it into the rat cardiac myocytes cultured in vitro.Methods A pair of primers were designed,and full length MAP4 DNA was cloned from rat total mRNA by PCR.The PCR product was double-digested with restriction endonucleases NheⅠ and NocⅠ,and inserted orientationally into pShuttle2.The plasmid of pShuttle2-MAP4 was double-digested with restriction endonucleases NheⅠ and NocⅠ,and inserted BD Adeno-X~(TM) Virul DNA,named pAd2-MAP4.The non-recombinant adenovirus was screened out with PacⅠ, pAd2-MAP4 was linerized with SwaⅠ,and the recombinant virus genome was transfected into HEK293 cell line for packaging and amplification of Ad-MAP4 virus.The recombinant adenovirus was transfected into rat cardiac myocytes and MAP4 was identified by immunohistochemistry.Results The recombinant adenovirus-MAP4 was constructed successfully and the titer was about 2.3?10~(8) pfu/ml.The expression of MAP4 was enhanced at 48 h after the transfection.Conclusion We have successfully constructed a recombinant adenovirus Ad-MAP4 that has enforced the expression of MAP4 in vivo.
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Objective To investigate whether microtubule disassembly plays an important role in the pathogenesis of the opening of mitochondria permeability transition pore (MPTP) in hypoxic cardiomyocytes and the decrease of its activity, resulting in its hypoxic injury. Methods Neonatal rat cardiomyocytes in primary culture were randomized as normoxia group (A), hypoxic group (B), normoxia treated with microtubule destabilizing agent (Colchicine) group (C), hypoxia treated with microtubule stabilizing agent (Taxol) group (D). At 0.5, 1, 3, 6, 12 h after treatment, polymeric tubulin was detected by immunofluorescence and Western blotting, mitochondria permeability transition pore (MPTP) open by coloading with calcein AM and cobalt chloride, and the activity of cells by measuring the mitochondrial-dependent reduction of MTT to formazan. Results Early microtubule disassembly, MPTP open and activity decrease of cardiomyocytes in both groups B and C were observed at 0.5 h after treatment. These phenomena all became more and more significant with the prolongation of treatment. However, microtubule disassembly, MPTP open and activity decrease of cardiomyocytes of group D were significantly lower than those of group B. Conclusion Microtubule disassembly happened at 0.5 h after hypoxic treatment. Microtubule stabling agent Taxol and destabilizing agent Colchicine can regulate microtubule integrity efficiently. The microtubule damage plays an important role in the hypoxic injury of cardiomyocytes.
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Objective To investigate the protective effect of glycine (Gly) on hypoxic injury to murine cardiomyocytes and the mechanisms. Methods The survival rate of cardiomyocyte survival was detected by trypan blue exclusion and lactate dehydrogenase (LDH) with an ultraviolet spectrophotometer. Ca 2+ changes in the cardiomyocytes were detected by laser confocal microscopy. Results Glycine markedly improved the survival rate of cardiomyocytes and decreased the release of LDH in cardiomyocytes after hypoxia. The protective effect was in a dose-dependent manner. Glycine could also block calcium overload after hypoxia. Conclusion Glycine has the protective effect on cardiomyocytes through the improvement of survival rate, decrease of LDH release, and blockage of calcium overload after hypoxia.
